Phospholipid-coated microcrystals: injectable formulations of water-insoluble drugs

ABSTRACT

Water-insoluble drugs are rendered injectable by formulation as aqueous suspensions of phospholipid-coated microcrystals. The crystalline drug is reduced to 50 nm to 10 μm dimensions by sonication or other processes inducing high shear in the presence of phospholipid or other membrane-forming amphipathic lipid. The membrane-forming lipid stabilizes the microcrystal by both hydrophobic and hydrophilic interactions, coating and enveloping it and thus protecting it from coalescence, and rendering the drug substance in solid form less irritating to tissue. Additional protection against coalescence is obtained by a secondary coating by additional membrane-forming lipid in vesicular form associated with and surrounding but not enveloping the lipid-encapsulated drug particles. Tissue-compatible formulations containing drug in concentrations up to 40% (w/v) are described. The preparations can be injected intra-lesionally and in numerous other sites, including intra-venous, intra-arterial, intra-muscular, intra-dermal, etc. The disclosure describes examples of formulations and pharmacokinetic data with antibiotics, anthelmintic drugs, anti-inflammatory drugs, local and general anesthetics, and biologicals.

This invention relates to an injectable delivery form enabling theinjection of high concentrations of water-insoluble drugs into amammalian host and affording sustained release of the injected drug. Thepresent invention shows that crystalline water-insoluble drugs can bereduced to submicron dimensions and suspended in aqueous media at highconcentration in a pharmaceutically elegant injectable form by coatingwith a membrane-forming lipid. The coating is generally a phospholipidbut can be made from any membrane-forming lipid. The microcrystal iscoated by a layer of membrane-forming lipid which stabilizes themicrocrystal by both hydrophobic and hydrophilic interaction. The fattyacyl chains of the phospholipid stabilize the microcrystal byhydrophobic interaction and the polar head groups of the phospholipidstabilize the coated-microcrystal through their interaction with solventwater. The coated microcrystal can be further stabilized by envelopmentby the lipid in bilayer form, and by the inclusion of excessmembrane-forming lipid in the suspending medium in the form of vesicles.The preparation is tissue-compatible and gives sustained release uponintra-muscular (IM), subcutaneous (Sub-Q), intra-dermal injection orinjections into other confined tissues or spaces (intra-peritoneal,intra-articular, epidural, etc.). The preparation is capable of givingrapid release when injected into the blood, a large and less confinedcompartment in which it experiences rapid dilution. The preparation canbe injected intra-lesionally to produce high local doses withoutinvolving the rest of the system. The invention is distinguished fromexisting drug delivery systems by its injectability, itstissue-compatibility, its small particle size, its high payload, itssyringability and stability in storage, its use of phospholipid as thesole coating material, and its non-antigenicity.

BACKGROUND OF INVENTION

Water-soluble drugs are readily injectable. Water-insoluble drugs arenot. For water-insoluble (or oil-soluble) drugs the creation ofinjectable forms represents a substantial problem. The Pharmacopeacontains many examples of water-insoluble drugs which must be takenorally because no adequate injectable form exists for them. Present artis limited in terms of the drug concentration and total volume which canbe injected. Its application is limited by problems of local irritation,tissue destruction, etc. (in IM injection) and thrombophlebitis,thromboembolism, pulmonary capillary blockage, etc. (in IV injection).

As a preliminary to discussing prior art, it is useful to consider thecriteria required of injectable preparations. The following criteria canbe extracted from current clinical practice and from general guidelinesused by the U.S. Food and Drug Administration in licensing newinjectable products.

A. The preparation and its vehicle must be tissue-compatible: Thisrequirement is equally important for injection into tissues and into thecirculation. Injection of a deleterious agent into muscle can causepain, irritation, tissue destruction, cellular reactions, fibrosis orpurulent reactions. Injection of a deleterious agent into thecirculation can result in thrombophlebitis, including damage to theartery or vein, clot formation in the artery or vein and blockage of thecirculation to the tissue or the lungs. As described below,solubilization strategies involving the use of organic solvents, extremepH and detergents are severely limited by these problems.

B. The formulation must not contain particles of diameter >10 um:Particles with dimensions greater than 10 um will block bloodcapillaries. If administered intra-arterially (IA), they will lodge inthe capillaries of the tissue, causing local ischemia. If administeredIV, they will lodge in the lung capillaries and cause respiratorydistress. For reasons of safety, the <10 um criterion must be met forother intended routes of injection (e.g., intra-muscular, IM) due to thedanger of inadvertent IV or IA injection. As described below, most ofthe controlled release technology directed at oral dosing isinapplicable to injectable forms because it fails to meet thiscriterion.

C. The formulation must allow injection of sufficient quantities ofdrug: The formulation must carry the drug at high concentration. As anexample, if the highest concentration available for a drug is 2% (w/v)or 20 mg/ml and the largest practical volume for an IM injection intoman is 5 ml, then a single injection can supply only 100 mg of the drug.If the drug is sufficiently potent, this will present no problem.However, there are many examples in which 1-2 gm of drug must beintroduced into the body. This would require either a 10- or 20-foldlarger volume (impractical or impossible) or 10-20 times theconcentration (heretofore unachievable).

D. The formulation must not rely on constituents which may elicit anallergic response: This is a particular problem for injections into theskin and muscle. Repeated injections of foreign proteins ormacromolecules can elicit an immune response. Much of the present art incontrolled release relies on "plastics", crosslinked serum albumin, orpolymers such as poly (D,L) lactic acid.

E. To be generally useful, the delivery system must have a high"payload": Payload can be defined as the ratio of weight drug deliveredto weight of carrier, or encapsulating substance. For example, if adelivery system uses 10 gm of wax or polymer to encapsulate 2 gm ofdrug, then its payload is 0.2. Delivery systems with low payload willrequire large amounts of encapsulating substance. The ability of thetissue or vascular compartment to metabolize or remove this substance,however benign, will limit the amount of drug which can be given.

F. The formulation must be stable, grossly homogeneous, syringable andpharmaceutically elegant and must maintain these properties for areasonable shelf life: The phospholipid-coated microcrystal disclosed inthis specification is unique in that it satisfies all of these criteria.It is also unique in that, while satisfying the above criteria, itenables water-insoluble drugs to be injected at high concentrations ashigh as 40% (w/v).

PRIOR ART

Prior art can be considered in terms of both current pharmaceutical andclinical practice, and in terms of the patent and scientific literature.The survey below shows that none of the existing systems fulfills all ofthe 6 criteria described above for an injectable form of awater-insoluble drug.

COMMERCIALLY-AVAILABLE FORMS

Medications falling into this category can be definitively surveyed byreference to the Physicians' Desk Reference (PDR, from Medical EconomicsCompany, Inc., Oradell, N.J.) which describes all licensed products inthe U.S. One approach to the problem is to render the drug water-solubleby ionizing it using non-physiological pH. As an example, thiopental issupplied as a sodium salt, which upon addition of sterile water, makesan alkaline solution of the drug at 0.2-5% which can be IV-injected.Listed adverse reactions include venous thrombosis or phlebitisextending from the site of injection (Abbott, 1988 PDR, p. 556-559).

Another approach, applicable to IM injection only, has been to inject asolution of compound in vegetable oil. Although the triglycerides invegetable oil are tissue compatible, bulk oil is not readily absorbed ormetabolized by the body. Oil boluses become "walled off" by growth ofencapsulation tissue and can persist for months. Due to problems withoil granuloma and serious risks associated with inadvertent IV or IAinjection, this approach is largely superseded. A remaining example is a7.05% solution of haloperidol decanoate in sesame oil (McNeil, PDR, pp.1240-1241).

Another approach to the problem is to solublize the drug in an organicsolvent. As an example, diazepam (Valium®) is solublized to aconcentration of 5 mg/ml (0.5%) in a solution of 40% propylene glycol,10% ethyl alcohol, water and preservatives. For IV use, warnings aregiven to reduce the possibility of venous thrombosis, phlebitis, localirritation, etc. which are reported under adverse reaction. Thepreparation is not stable to dilution in water (Roche, 1988 PDR, pp.1764-1766.

Due to local reactions of organic solvents, their human use is fairlyrestricted. This is not the case in veterinary use in food animals inwhich high drug concentrations are required due to the volumelimitations of the syringe (20 ml) and other practical considerations.An example is a commercial 10% (w/v) alkalinized solution ofoxytetracycline in propylene glycol for IM injection in cattle. Thisproduces pain on injection and local damage.

A different approach is to solublize or suspend the drug with non-ionicdetergent. An example of this is a cortisone acetate suspension,consisting of 25 mg/ml or 50 mg/ml cortisone acetate, 4 mg/mlpolysorbate 80, 5 mg/ml sodium carboxymethylcellulose and preservativesin isotonic saline (Merck Sharp & Dohme, 1988 PDR, pp. 1297-1299).Indications are for intra-muscular use only, when oral therapy is notfeasible. In some cases an organic solvent and detergent are combined. Apre-anesthetic sedative product containing 2 mg/ml or 4 mg/ml lorazepamand 0.18 ml/ml polyethylene glycol 400 (non-ionic detergent) inpropylene glycol is available for IM injection or IV injection afterdilution. It is reported to cause pain and burning with 17% incidencewith IM injection (Wyeth, 1988 PDR, pp. 2258-2259).

FORMS DESCRIBED IN THE PATENT AND SCIENTIFIC LITERATURE

Many of the systems in this category make use of fatty acids, "fats",phospholipids, and non-ionic surfactants. For the reader's conveniencethe chemical and physical nature of these substances will be describedbriefly in the remainder of this paragraph. Alkali cation salts of fattyacids are "soaps" which tend to form micelles of ≦5 nm diameter whenmixed with water. They are also capable of coating larger hydrophobicstructures. Esterification of a fatty acid with glycerol (CH₂OH--CHOH--CH₂ --OH) produces monoglycerides (e.g. glycerol monooleate)are either oils or solids in the pure state, depending on their fattyacid chain lengths and temperature. Under some circumstances they arecapable of forming or participating in membranous structures in thepresence of excess water. With esterification of two or three OHpositions of the glycerol (diglycerides and triglycerides, respectively)this property is lost. Triglycerides are major constituents of "fat" andvegetable oil. They do not form membranes or participate in membranestructures. Phospholipids are 1,2 diacyl esters of glycerol, with the 3position esterified with phosphate. They are the major building block ofbiological membranes, and are very tissue compatible. An important andabundant example is lecithin (phosphatidylcholine), in which thephosphate group is esterified with choline, producing a zwitterionicpolar head group. In the presence of excess water, phospholipids formmembranes of bimolecular thickness. The polar head groups are orientedto the water; the fatty acyl chains form a palisade structure, withtheir ends abutting in the center of the membrane. Non-ionic detergents(or surfactants) used in drug formulations are high molecular weightpolymers of alternating hydrophobic and hydrophilic segments. Anoften-used and cited example is polyethylene glycol, which has thestructure H(O--CH₂ --CH₂)_(n) OH. Non-ionic detergents are capable ofcoating and solublizing hydrophobic oils and solids in aqueous media.They do not form membranes. They lyse biological membranes and are thusnot tissue compatible.

Wretlind et al. (U.S. Pat. No. 4,073,943, 1978) described the use of fatemulsions, of the type used for intravenous feeding, as a carrier forthe intra-venous administration of water-insoluble drugs. The describedexamples typically contained 0.5%-3.75% drug and 20% (w/v) vegetable oilas carrier. The drug/oil ratios (or payload) were typically 0.05, andranged between 0.013 and 0.375. This essentially limits the fat emulsionto intravascular delivery and limits the total amount of drug which canbe administered each day (cf. Criterion E, above). This restrictionapplies to other patents involving IV fat emulsions (Mizushima et al.,U.S. Pat. No. 4,613,505, 1986; List et al., U.S. Pat. No. 4,801,455,1989). The system is the basis of Diazemuls®, an IV-injectable productof Pharmacia of Canada, containing 0.5% (w/v) diazepam, 15% (w/v)soybean oil and other constituents.

Haynes (U.S. Pat. No. 4,725,442, 1988) described injectable aqueoussuspensions of phospholipid coated microdroplets of water-insolubledrugs. The drugs were themselves oils (e.g. inhalation anesthetics) orwere dissolved in a pharmacologically-acceptable oil. The presentinvention offers improved payload for crystalline, water-insolubledrugs.

Liposomes, vesicles formed from membrane-forming phospholipids such aslecithin, were first described by Bangham, Standish & Watkins (in J.Mol. Biol. 13:238, 1965). That publication proposed the bilayerstructure described above. Homogenization of phospholipid in waterproduces multi-lamellar phospholipid vesicles consisting of concentricbilayer membranes. Sonication produces small unilamellar phospholipidvesicles as described by Haung (in Biochem. 8:344, 1969). Liposomes havethe ability to entrap polar and highly-charged molecules in theiraqueous interiors. The fact that liposomes are a non-antigenic deliverysystem (Criterion D) is widely appreciated. There are numerous patentsdirected to their properties of entrapment and delivery of water-solubledrugs. In a smaller number of patents, liposomes have been shown capableof incorporating oil-soluble drugs, but the payloads are low. Asexamples Schrank (U.S. Pat. No. 4,411,894, 1983) with payload=0.01-0.033gm diazepam or flunitrazepan/gm phospholipid, Mezei & Nugent (U.S. Pat.No. 4,485,054, 1984) with payload=0.18-0.20 gm progesterone/gmphospholipid plus cholesterol, Dingle et al. (U.S. Pat. No. 4,427,649,1984) with payload =0.10-0.125 gm fatty acylated steroid/gmphospholipid, or Abra & Szoka (U.S. Pat. No. 4,766,046, 1988) withpayload=0.03-0.07 mole amphotericin/mole phospholipid. With theexception of diazepam and flunitrazepan, all of the above drugs aremembrane active agents which are expected to incorporate into membranes.That they can not incorporate at levels above 0.2 gm/gm is indicative ofan upper limit of the degree of loading and disruption of the palisadestructure of the phospholipids in the bilayer.

Sears and Yesair (U.S. Pat. No. 4,298,594, 1981) described incorporationof adriamycin, imidocarb and estradiol decanoate into "microreservoirs",described therein as a mixture of vesicular and non-vesicularstructures, consisting of phospholipid and a much lower concentration ofcholesterol esters. Drug concentrations were low 0.002%-0.32%, and thepayloads were low (0.00066-0.027 gm drug/gm phospholipid pluscholesterol ester.)

The present invention makes use of phospholipid to suspendwater-insoluble drugs, but in a completely different way than describedin the above liposome patents. Rather than attempting to dissolve thedrug in the lipid bilayer, my invention retains the drug in crystallineform and uses the phospholipid to coat the crystal. The phospholipidvesicle is not an integral part of the lecithin-coated microcrystal.

There are numerous examples of drugs coated with wax or "lipoidalmaterials". However, the examples are directed at oral administration,and in some cases topical administration. For example tristearin(triglyceride of glycerol and stearic acid) is a common constituent oftablets. It is a "fat" with the physical form of a powder or wax atnormal temperature (Merck Index, 10th Edition, Merck & Co., Rahway,N.J., 1983, p. 1293). In some cases the coating is melted on (Augart,U.S. Pat. No. 4,483,847, 1984; Kondo, U.S. Pat. No. 4,102,806, 1978).Various improvements of this technique have been patented: Ohkawara etal., U.S. Pat. No. 4,675,236, 1987), again as oral administration forms.A patent of Ghyczy et al. (U.S. Pat. No. 4,378,354, 1983) describes"pills" of non-steroidal anti-inflammatory drugs containingphospholipids.

Forms involving especially small crystals have been termedmicrocapsules. The microcapsule (exemplified by Morishita et al., U.S.Pat. No. 3,960,757, 1976) is generally a 30-1,200 u capsule of drugcoated usually with a water-insoluble material such as ethyl cellulose(Alam & Eichel, U.S. Pat. No. 4,316,884, 1982), wax or insoluble salt ofa fatty acid. The sizes of microcapsules are often described in terms of"mesh", with typical ranges of 50-200 mesh (300 um to 75 um), with someexamples at least 10 u up to 5 mm. They are used in tablets or to fillcapsules. Wax is generally recognized to be water-repelling, and waxycoatings do not lend themselves to aqueous suspensions. Wax-coatedmicrocapsules are primarily directed towards oral use which does notrequire stable suspensions in aqueous media, injectability andcompatibility with tissue, or small size (Criteria A, B, and F, above).

The patent literature contains numerous examples of preparationsdirected at topical use containing crystalline drugs mixed withglycerol-lipids such as glyceryl monooleate (Reller, U.S. Pat. No.4,219,548, 1988). The cosmetic literature also offers numerous examplesof creams of high water content (cf. Nieper & Melsungen, U.S. Pat. No.3,274,063, 1966) to which crystalline drugs can be mixed. These creamscan not be considered injectable because they constitute a self-adherentmass which does not dissociate to give particles small enough to passthrough blood capillaries (Criterion B). Additionally, the tissue- andblood compatibility of the surfaces presented by these topicalpreparations has not been demonstrated (Criterion A). A more recentexample not meeting these criteria and Criterion F is the U.S. Patent ofMezei (U.S. Pat. No. 4,761,228, 1988). It describes the topicalapplication of minoxidil and econazole nitrate to skin at concentrationsof 1.2-3%, in a preparation containing multi-lamellar lecithin vesicles.The preparation contained the drug in crystalline form. Some of the drugcrystals were within the vesicles, and the others were not associatedwith them. Multilamellar vesicle sizes were between 1 um and 15 um;crystal sizes were between 1 um and 20 um. The formulation was preparedby codissolution in organic solvents, followed by evaporation, additionof aqueous media and shaking. No information on immediate- or long-termstability was given.

The microsphere consisting of drug incorporated into 1-200 um diameterspheres of heat-hardened serum albumin, with the precipitated drugincorporated therein, was described by Zolle (U.S. Pat. No. 3,937,668,1976). In some cases it has been described as an injectable form. Widderand Senyei (U.S. Pat. No. 4,345,588, 1982) described the IV injection ofalbumin microspheres consisting of drug, serum albumin and Fe₃ O₄ powderin a ratio of 10:125:36. The albumin is crosslinked by formaledhyde.Particle diameter was 10 um. However, the antigenicity question was notaddressed. Mosier (U.S. Pat. No. 4,492,720, 1985) described 50 um-350 umdiameter spheres designed for "chemoembolization" via intra-arterialdelivery. The microspheres consisted of water-soluble drugs bound in amatrix of hydrogenated lard or vegetable oil and stabilized bydetergents. Morris (U.S. Pat. No. 4,331,654, 1982) described alyophilized preparation of <3 um diameter magnetically-localizablemicrospheres consisting of a core of magnetite (Fe₃ O₄) coated with asolidified mixture of fatty acid and non-ionic detergent, and containinglecithin as a minor constituent. He suggested, but did not demonstrate,that drugs could be incorporated at a weight ratio of 0.02-0.15 gmdrug/gm fatty acid. Blood and tissue compatibility, resuspendability andstability of suspensions were not discussed.

A classification which fulfills the size criterion for injectability isthe nanoparticle. Oppenheim et al. (U.S. Pat. No. 4,107,288, 1978)described particles of 120-660 nm diameter consisting ofglutaraldehyde-fixed gelatin. These included phenobarbitone at lowpayload 0.0067-0.0979 gm drug/gm gelatin (denatured collagen).Antigenicity and blood compatibility were not described. Couvreur et al.(U.S. Pat. No. 4,329,332, 1982) described ≦500 nm diameter particles ofalkyl-cyano-acrylate incorporating drug at low payload 0.0012-0.062, andapparently stabilized by detergent. No information was given on blood ortissue compatibility or stability of the suspension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the phospholipid-coatedmicrocrystal. The symbol O== is a phospholipid (O is polar head; ═ isthe pair of fatty acyl chains). Diameter is 0.5 um (range 0.05-10 um).

FIG. 2 presents drawings of a field observed with a fluorescentmicroscope when the 20% (w/v) oxytetracycline, 20% (w/v) egg lecithinmicrocrystal preparation, doped with Nile Red, was spread on a slide.White indicates high fluorescent intensity.

FIG. 2B shows the pattern of oxytetracycline fluorescence. The smallerparticles are approx. 0.2 um diameter.

FIG. 2A is of the identical field excited at the Nile Red wavelength.This fluorescence shows the distribution of the lecithin in thepreparation.

FIG. 3 shows the percent of oxytetracycline remaining in the leg muscleof rats (n=4) after injection of 0.1 ml of 20% OTC in microcrystalineform coated with 5% egg lecithin. The data for days 1-7 post-injectionare compared with results for the same quantity of OTC injected as acommercial 2-methyl-pyrolidone solution.

FIG. 4 shows the levels of OTC in central arterial blood in theexperiment of FIG. 3.

FIG. 5 shows the blood levels of OTC in calf after intra-muscularinjection of 20% (w/v) OTC (10% (w/v) lecithin-coated) microcrystals.

FIG. 6 shows the time course of protection against edema by 5 mgindomethacin injected intra-muscularly as lecithin-coated microcrystals,compared with an equal dose injected as an alkaline solution.

FIG. 7 shows typical time courses of the level anesthesia in ratsmeasured as vocalization threshold (mAmp) to intradermal electricalstimuli after intra-venous injection of lecithin-coated alphaxalonemicrocrystals.

FIG. 8 shows the time course of anesthesia in the human skin (pin prick)achieved with lecithin-coated 20% (w/v) tetracaine hydroiodic acidmicrocrystals 8A, compared with tetracaine-HCl solutions 8B.

DESCRIPTION OF THE INVENTION

My invention provides a means for creating injectable, tissue-compatiblesuspensions of water-insoluble drugs at high concentrations. This allowsthe parenteral (injection) administration of drugs. It is generallyapplicable to any water-insoluble drug which is in the crystalline stateat 37° C. Formulation as a phospholipid-coated microcrystal enables thedrug to be injected or otherwise parenterally administered. Theformulation is unique in satisfying all of the 6 criteria(tissue-compatibility, ≦10 um size, injectable quantity,non-antigenicity, payload, and physical stability) for amaximally-useful injectable form. The relationship between themicrocrystal and the coating phospholipid is depicted schematically inFIG. 1. Central to my invention is the use of the amphipathic oramphiphilic properties of phospholipids in general and lecithin inparticular. Webster's Medical Desk Dictionary (Merrian-Webster Inc.,Springfield, Mass., 1986) defines amphipathic/amphiphilic as " . . .consisting of molecules having a polar water-soluble terminal groupattached to a water-insoluble hydrocarbon chain." In FIG. 1, the polarhead group of the phospholipid is denoted by circles and the hydrophobichydrocarbon chains are denoted by "sticks". Many substances areamphipathic, including soaps, surfactants and detergents. Unique to myinvention is the use of phospholipids to shield the hydrophobic surfaceof the crystalline drug and to provide additional membranous barriersagainst reassociation of the crystals. Other amphipathic molecules suchas soaps, surfactants and detergents are unable to provide such stableand tissue-compatible structures. Also unique to my invention is themeans of forming these stable phospholipid-coated microcrystallinestructures. This is described below.

SIZE REDUCTION AND PRIMARY COATING

As described herein, the crystalline drug substance is reduced to <10 umor submicron dimensions in an aqueous medium by sonication or othertreatments involving high shear. Lecithin (or other membrane forminglipid), present during the sonication, is itself broken into highlyreactive fragments with exposed hydrophobic surfaces. These fragmentscoat and envelop the submicron crystals creating a primary coating. Arequirement for this process is that the lecithin and drug be presenttogether during the sonication or alternative high-energy dispersingprocess. (Sonication of drug crystals, followed by rapid mixing ofpre-formed phospholipid vesicles does not give stable submicron aqueoussuspensions of the drug.) The subsection entitled "Methods ofPreparation" specifies alternative methods involving inflightevaporative coating and solvent dilution. The common aspect of all ofthese preparative methods is that the fatty acyl chains of thephospholipid must have direct access to the microcrystal during thecoating process.

In my invention, the amphipathic properties of the phospholipid satisfyboth the hydrophilic properties of water and the hydrophobic propertiesof the crystal surface. Also, the phospholipid membrane surface servesas a stationary barrier to reformation of macroscopic crystals. A seconduseful property of the primary coating is modification of the rate ofthe dissolution process. Possible structural features of thephospholipid-microcrystal interaction are schematized in FIG. 1.

SECONDARY COATING: PERIPHERAL PHOSPHOLIPID

In addition to making use of lecithin and other membrane-forming lipidsas a coating and enveloping material, my invention makes novel use ofmembrane-forming lipids as mechanical buffers, organizers of aqueousvolume and retardants of recrystallization of the drug. This is achievedby excess phospholipid in the form of unilamellar and multi-lamellarphospholipid vesicles which form a secondary coating of the suspendedmicrocrystal. Predominently unilamellar vesicles are formed as abyproduct of the sonication and primary coating process. Their retentionin the preparation was found to improve the long-term stability of theformulation. Also, preformed multi-lamellar vesicles (made byhomogenization) or uni-lamellar vesicles can be added to the preparationto improve its stability or pharmacokinetics (Example 5). The secondarycoating is loosely attached to the coated microcrystal. Peripheralvesicles associate with and dissociate continuously in the preparation.The secondary coating can be removed by repeated centrifugation andresuspension of the preparation (Examples 3 and 11).

Peripheral vesicles forming a secondary coating stabilize thepreparation. While not wishing to be bound to any particular theory ormode of action, detailed consideration has suggested the followingmechanisms:

They act as volume buffers interposed between the primary-coatedmicrocrystals. The crystalline and microcrystalline drugs are often moredense than the phospholipid which is, in turn, more dense than water.Thus they will tend to settle under the influence of gravity and willexperience greater long-range interactions (van der Waals attraction)than the other two constituents. The secondary coating increases thedistance of closest approach of the microcrystalline drug cores, therebydecreasing the van der Waals attraction. It is possible that part of thedriving force for the secondary coating is van der Waals attractionbetween the primary-coated microcrystal and the phospholipid vesicle.Phospholipids (notably lecithin) are ideal as the primary and secondarycoating because they are strongly hydrated and engage in well-documentedshort-range repulsive interactions which make them very resistant toaggregation and fusion.

When peripheral phospholipid is present at 20% (w/v), the majority ofthe aqueous volume of the preparation is enclosed within phospholipidmembranes. This serves as a topological barrier to recrystallization ofthe drug in a preparation during long-term storage. Re-formed crystalscan not be larger than diameter vesicle or distance between them, bothof which can be kept small.

PHYSICAL CHARACTERISTICS OF FORMULATION

Sonication is most conveniently carried out with the drug atconcentrations of 5% (w/v) or less and the membrane-forming lipid at 5%or greater. This results in a syringable suspension of coatedmicrocrystals of predominantly sub-micron dimensions, with the particlesexhibiting Brownian motion (Examples 2, 3 and 11). Over a period of 1-2days the microcrystals settle creating a distinct zone in which the drugconcentration is 20-40% (w/v). The final concentration and volume aredependent on the choice of the drug and upon the peripheral phospholipidconcentration. In most preparations the bottom zone is resuspendablewith inversion to give a homogeneous and syringable suspension, evenafter a period of months. For preparations in which this was not thecase, resuspendability was obtained by increasing the peripheralphospholipid concentration.

The slow sedimentation process can be used as a means of concentratingthe preparation. Removal of the volume above the sedimentation zoneafter 1-2 days results in preparations in which the drug is at 20-40%(w/v). Long-term storage results in no further settling. Thepreparations remain homogeneous, syringable and pharmaceuticallyacceptable for many months (cf. Examples). Microscopic examination ofthese preparations reveals separated micron and sub-micron diametercrystals of the drug. The volume between these drug microcrystals isalmost completely filled with phospholipid vesicles, visualized by NileRed staining (cf. Examples 2, 3 and 11). In this concentrated form, thedrug microcrystals exhibit only restricted Brownian Motion. Undermicroscopic observation they are not observed to change position inrelation to eachother. They vibrate or "dance in place" about theircentral position. This partial restriction of motion is probably animportant factor in the long-term stability of the preparation. Whenstored, concentrated preparations are diluted many thousand-fold intodrug-saturated water, the microcrystals retain their micron orsub-micron size.

MODES OF ADMINISTRATION

As noted above, the primary utility of the coated microcrystal is itsinjectability. Applicable injection sites are any tissue or body cavity.They include but not limited to intra-venous (IV), intra-arterial (IA),intra-muscular (IM), intra-dermal, sub-cutaneous (Sub-Q),intra-articular, cerebro-spinal, epi-dural, intra-costal,intra-peritoneal, intra-tumor, intra-bladder, intra-lesional,sub-conjunctival, etc. In addition, the phospholipid coating andsubmicron size of the preparation may prove to have advantages for oraluse, both as an aqueous suspension and as a lyophilized product.Similarly, the aqueous suspension may show advantages for topicalapplication, instillation into the eye. The preparation can deliverdrugs by the inhalation route, in the form of either an aqueoussuspension or a lyophilized powder. It is also likely that thepreparation will be useful for administration of pesticides and increating high value biocompatible products, as exemplified by suspensionof drugs in drinking water (Example 15).

RATE OF RELEASE

The most important determinant of the rate of release of the drug is thechoice of injection site. If the formulation is injected intravenously,it can be released from the microcrystal quite rapidly. If theformulation is injected at high volume into a confined space such asmuscle, the net rate of release can be exceedingly slow. Theintra-venous case will be considered first.

The blood is a fluid medium which is capable of diluting the preparation1,000,000-fold within approx. 1 min. When a concentrated lecithin-coatedmicrocrystal preparation is diluted in blood, the individualmicrocrystals, initially in an environment consisting of other coatedmicrocrystals, peripheral lipid and drug-saturated water, aretransferred to an environment consisting of serum proteins, serumlipoproteins and cellular blood elements. My in vitro fractionationexperiments (Examples 3 and 11) suggest that the secondary coating willbe rapidly lost. All of the blood elements are capable of bindinglipophilic molecules and will do so as rapidly as the microcrystal candissolve. In cases where the drug is sufficiently water soluble,dissolution into the aqueous portion of the blood is sufficient todistribute the drug. When water-solubility is insufficient, a continuousprocess of dissolution and binding of the drug to blood elements servesto remove the drug from the microcrystals. The rate of dissolution ofthe microcrystal will depend upon the thickness and stability of itsprimary coating, the water-solubility of the drug and otherphysico-chemical parameters. Example 10 shows that the anestheticalfaxalone can leave the microcrystal and enter the brain within 10 secof its IV injection. It is possible to reduce the rate of release afterIV administration by variation of the thickness of the primary coatingor by inclusion of small quantities of water-insoluble oil (such asvitamin E) in the preparation.

With injection into a tissue such as muscle, the preparation does notundergo rapid dilution. It generally remains in the initial elements ofvolume created by the injection. These are generally macroscopic andthere is little flow or agitation. Diffusion the drug out of this volumeis slow because of the relatively large distance involved and is furtherslowed by the low water solubility of the drug. The larger the injectedvolume and the lower the water solubility, the slower will be the rateof removal of the drug (Example 7). In the extreme, the release processcan require upwards of 14 days. For high and fixed volumes and drugconcentrations, the rate of removal can be increased by incorporation ofhypertonic glucose or carboxycellulose in the vesicles of the secondarycoating. This resists the mechanical pressure of the tissue which tendsto solidify the injected preparation (Example 5). IM injection is usefulto create a depot of drug and to obtain sustained release to the bloodover a period of days. Injection directly into the target tissue orlesion is useful because it achieves high and sustained concentrationsof the drug at the site where it is needed without involving the rest ofthe system.

METHODS OF PREPARATION

1. Sonication: The sonication process reduces the size ofsupra-molecular structures by the process of cavitation. The processcreates small empty volumes which collapse, propelling material togetherat high speed, resulting in shattering and sheer. This simultaneouslybreaks up the drug crystals and phospholipid lamellae into submicronfragments. The phospholipid membranes are shattered in directions bothparallel and perpendicular to their planes, yielding surfaces which cancoat the hydrophobic surface of the microcrystal, an which can rejoin toenvelope it, respectively. Thus the phospholipid concentration must beadequate for the rate of coating and enveloping to exceed the rate ofrejoining of broken crystals. I have observed that sonication usuallyworks well when the drug concentration is 5% (w/v) or lower and when thephospholipid concentration is 5% or greater. The role of the secondarycoating of phospholipid vesicles has been described above.

It does not work well if the drug and lipid are sonicated separately andadded together. In fact, in the absence of a membrane-formingphospholipid one seldom succeeds in reducing the drug to sub-micron sizefor even a short time. Sonicated or homogenized lipid can be added toalready-prepared coated microcrystals to increase or modify theirperipheral lipid content. As described above, the preparation can beconcentrated to 20%-40% (w/v) by allowing it to settle.

The product can be put into dry form by lyophilization to yield a powderwhich can be later reconstituted (Example 6). This is useful when thelong-term chemical stability of the to-be-encapsulated drug in anaqueous environment is poor.

2. Methods involving high pressure and shear: The crystalline drug andphospholipid are pre-mixed by high-speed homogenization (as with WaringBlender. Further size reduction and coating can be accomplished by theprocess of Microfluidization® (Microfluidics Corp., Newton Mass 02164).The process relies on high shear created by collision of opposing jetsof liquid. The apparatus is described by Mayhew et al. in Biochim.Biophys Acta 775: 169-174, 1984. An alternative is high pressurehomogenization by means of the French Pressure Cell or "French Press"(SLM Instruments, Urbana, Ill.). In this process, the sample is forcedat high pressure and high shear through a narrow orifice and undergoesrapid decompression to atmospheric pressure. Other details are as in #1above.

3. Sonication or high shear in volatile organic solvents: Microcrystalscan be prepared by suspending the crystalline drug and themembrane-forming lipid in a volatile non-polar solvent in which the drugis poorly soluble (dichlorodifluoromethane or dichlorotetrafluroethaneor Freon (e.g. trichlorotrifluorethane, c.f. Examples 6 and 8). Thesuspension is reduced in size by sonication or high-speed shear by themethods described above (#1 or #2). The solvent is removed byevaporation. The resulting powder can be stored for later reconstitutionwith water or can be reconstituted immediately.

4. Size reduction in air: Drug crystals can also be reduced in size byhigh speed impact in air. They can be subsequently coated by wettingwith a solution of phospholipid or glycerol lipid in a volatile solventcontaining lecithin, with the solvent removed by volatilization. Thepowdered product can be suspended in water. Alternatively, micronizedcrystals can be wetted by a water-miscible liquid dissolving lecithinand rapidly introduced into an aqueous medium.

5. In-flight crystallization: A solutions of the lipid and drug in avolatile solvent can be sprayed, with the solvent removed by evaporationwhile in flight. The microcrystals are collected and dried on a smoothsurface. The microcrystals can either be stored in the powdered form forlater reconstitution with water or can be reconstituted immediately.

6. Solvent dilution: Solutions of lipid and water-insoluble drug aremade using a water-miscible organic solvent (e.g. ethanol). Thesolutions are expressed into an aqueous medium with high agitation orsonication. The solvent dissolves in the water, leaving behind the drugin microcrystalline form coated with the lipid. The organic solvent canbe completely removed by filtration or by sedimentation of the coatedmicrocrystals and removal of the supernate.

SELECTION OF THE DRUG TO BE COATED

Any substantially water-insoluble drug which is in the crystalline orsolid state at temperatures of 37° C. is applicable. The drug shouldgenerally have a water solubility of <5 mg/ml at physiological pH(6.5-7.4). Use of drugs with higher water solubility is not precluded ifexperimentation shows minimal tendencies to reorganize into macroscopiccrystals during the desired shelf life. It is generally desirable tochoose a total drug concentration >4× the drug's water solubility, suchthat at least 80% of the drug is in the microcrystalline form. Thischoice takes advantage of the high payload and sustained-releasecharacteristics associated with the coated microcrystal. Finally, it ispreferred that the drug be intrinsically non-irritating. It is alsodesirable that the drug be chemically stable in a humid environment.Otherwise it may be necessary to produce lyophilized forms.

The most frequent examples are drugs which are water-insoluble but havemoderate-to-good oil solubility. However, oil solubility per se is not arequirement for incorporation into phospholipid-coated microcrystals.Many drugs which have tight crystal structures, high melting points arenot particularly water- or oil-soluble. These drugs can also benefitfrom phospholipid-coated microcrystal formulation. Similarly, it is notnecessary for the drug to be uncharged to be put into microcrystallineform. It is only necessary that the water solubility of the crystallineform of the drug be low.

RENDERING A WATER-SOLUBLE DRUG WATER-INSOLUBLE

It is possible to use an intrinsically water-soluble drug in myinvention providing that it can be rendered water-insoluble bycomplexation. For example an insoluble hydroiodic acid (HI) salt of thelocal anesthetic tetracaine is used to extend its duration of action5-fold (Example 12). If the drug is charged at physiological pH, it canoften be rendered insoluble by substituting a more lipophilic orstructured counter-ion. Examples for rendering a positively-charged drugless water soluble include complexation with 2-naphthylenesulfonate(napsylate), gluconate, 1,1' methylene bis (2-hydroxy-3-naphthalene)carboxylic acid (pamoate), tolylsulfonate (tosylate), methanesulfonate(mersylate), glucoheptanoate (gluceptate), bitartrate, polyglutamicacid, succinate, acetate, or behenate (anionic form of waxy fatty acid).In choosing fatty acyl anions it is advisable to select species witheither short chain lengths or very long chain lengths, such that thetendency of towards micellarization is minimized. In some casessubstitution with bromide, iodide, phosphate or nitrate is sufficient torender the drug less soluble. Examples for rendering anegatively-charged drug less water soluble include complexation withcalcium, magnesium or their 1:1 fatty acid salts, and with variousamines, including dibenzylethylenediamine (benzathine), N,N'(dihydroabietyl)ethylene diamine (hydrabamine) or polymers such aspolylysine. The choice of these counterions is made largely on anempirical basis, with stability of the derived crystals and theircompatibility with water being primary criteria. Since release of thedrug after dilution or injection can involve removal of both the chargedand the uncharged forms of both the drug and its counterion, thesesystems offer both complexity and diversity of kinetics. With sufficientstudy of the in vitro behavior of the phospholipid-coated microcrystalsmade from a number of these binary salt systems, and with judiciouschoice of the most promising examples, the desired in vivopharmacokinetics can be approximated.

Also, it is possible in some applications, to prepare microcrystals atmore extreme pH (4.0-6.4 or 7.5-10.0) in order to suppress ionizationand thus decrease the solubility of the drug. The allowable extremes ofpH in each particular case will be determined by the concentration ofthe drug, the number of acid or base equivalents which it carries, itsrate of dissolution and the size of the injected compartment and (interms of shelf life) the stability of the membrane-forming lipid.

SELECTION OF THE MEMBRANE-FORMING LIPIDS FOR COATING

The primary requirement is that the coating lipid be membrane-forming.This is satisfied by all lipids which, in the presence of excess water,make bilayer structures of the type which is well-documented forphospholipid vesicles or liposomes. This requirement is not satisfied byfatty acids, detergents, non-ionic surfactants (e.g. polyethyleneglycol) or triglycerides (vegetable oils, tristearin, "fats"). Asecondary requirement is that the lipid not have a proclivity forconverting into micellar structures. This excludes phospholipids ofshort chain length (6 or less) or lysolecithin (containing a singlefatty acyl chain). High stability of the coating material in membraneform is necessary to keep the drug material from rearranging intomacroscopic crystals. This is one reason why non-ionic surfactants donot work well for my intended purpose.

Useful examples of membrane-forming lipids are given below:

CLASS A: Primary phospholipids (usable in pure form) include thefollowing:

Lecithin (phosphatidyl choline)

Sphingomyelin

Synthetic zwitterionic phospholipids or phospholipid analogues

To this class belongs all phospholipids which spontaneously formmembranes when water is added. These phospholipids can be used in pureform to produce coated-microcrystals. Of all the phospholipids, lecithinis the most useful example because of its high availability and lowcost.

CLASS B: Phospholipids capable of calcium-dependent aggregation. Thesephospholipids include the following:

Phosphatidic acid

Phosphatidyl serine

Phosphatidyl inositol

Cardiolipin (diphosphatidyl glycerol)

Phosphatidyl glycerol

These lipids carry a negative charge at neutral pH. Preferably thesephospholipids can be mixed with lecithin to obtain negatively-chargedsurfaces which will give repulsion between particles. When introducedinto a medium containing 2 mM calcium (such as blood or interstitial),membranes containing these phospholipids are expected to show elevatedaggregation and higher reactivity with cell membranes. This can beuseful in causing the injected microcrystals to aggregate within thetissue, giving slower release rates. The usefulness of this class islimited by the high cost of these phospholipids, relative to lecithin.

CLASS C: Phosphatidyl ethanolamine promotes aggregation in acalcium-independent manner. It can be used in the pure form to coatmicrocrystals at pH 9. When the pH is brought to 7, as upon injectioninto blood or tissue the membranes become reactive, causing theparticles to aggregate and to attach to cell membranes. This can havethe useful property of slowing the release rate.

CLASS D: Cholesterol and steroids. These can not be used as a solecoating material: They do not form membranes in the pure state. They canbe added to the lecithin or other coating material to change its surfaceactivity, the "microviscosity" or distensibility of the coating. With asteroid hormone (estrogen, androgen, mineralo- or glucocorticoid), it ispossible to influence the local tissue response to the microcrystals aswell as influencing their physical disposition.

CLASS E: Semi-lipoidal molecules can be incorporated into thephospholipid or glycerol lipid membrane and change the surface activityof the microdroplet. Molecules included in this class are the following:

Stearylamine or other long-chained alkyl amines which can be primary,secondary, tertiary or quaternary substituted. These give themicrocrystal coating a positive charge and make them more reactive withcell membranes. Benzalkonium chloride is an aromatic example which isparticularly useful because it also functions as a preservative againstmicrobiological growth in the preparation.

Fatty acids. These can be incorporated at low concentrations (<0.02gm/gm phospholipid) to alter the phospholipid packing and reactivity.

CLASS F: Membrane-active agents, glycolipids and glycoproteins to modifysurface properties. Examples of membrane-active agents include nystatin,amphotericin B and gramicidin which are surface-active antibiotics.These have been shown to bind to the surfaces of phospholipid membranesand change their permeability. Glycolipids or glycoproteins could beincluded as a means of modifying surface reactivity. Likewise,antibodies can be coupled to membrane constituents to direct or retainthe microcrystal association with targeted cells or tissues.(Glycolipids, glycoproteins, and antibodies are classified as"biologicals". They would have to be screened for pyrogenicity,antigenicity etc. before use, and the process of gaining regulatoryapproval for such formulations would be more complex.)

CLASS G: Mono-glycerides. These are not phospholipids, but they havebeen shown capable of forming oriented monolayers and bilayers in thepresence of decane (Benz et al. Biochim. Biophys. Acta 394: 323-334,1975). They may thus prove have some use in coating for microcrystals.Examples of these lipids include, but are not limited to, the following:

1-monopalmitoyl-(rac)-glycerol (Monopalmitin)

1-monocaprylol-(rac)-glycerol (Monocaprylin)

1-monooleoyl-(rac)-glycerol (C18:1, cis-9) (Monoolein)

1-monostearyl-(rac)-glycerol (Monostearin)

COMMERCIALLY AVAILABLE MEMBRANE-FORMING LIPIDS

Several forms of lecithin are contemplated. As an example, egg lecithin(Sigma Chemical Co.) is used in all of the presented examples. It ispreferred for its low price and low degree of unsaturation. Lecithin isalso available from bovine heart. Soy bean lecithin is less expensive.It has a higher degree of unsaturation. Several synthetic varieties oflecithin are available which differ in chain length from 4 to 19 carbons(Supelco, Inc.). It is believed that lecithins with chain lengths in thebiological range (10-18) are useful in various applications. Unsaturatedlecithins (dioleoyl, dilinoleoyl; beta oleoyl; alpha-palmito betaoleoyl; alpha palmitoyl beta linoleoyl and alpha oleoyl beta palmitoyl)are also available. Diarachidonyl lecithin (highly unsaturated and aprostaglandin precursor) is also available.

Phosphatidic acid is available from egg or as synthetic compounds(dimyristoyl, dipalmitoyl or distearoyl, Calbiochem). Bovinephosphatidyl serine is available (Supelco or Calbiochem).

Phosphatidyl inositol is available from plant (Supelco) or bovine(Calbiochem) sources. Cardiolipin is available (Supelco) from bovine orbacterial sources. Phosphatidyl glycerol is available from bacterial(Supelco) courses or as synthetic compounds (dimyristoyl or dipalmitoyl;Calbiochem).

Phosphatidyl ethanolamine is available as egg, bacterial, bovine orplasmalogen (Supelco) or as synthetic compounds dioctadecanoyl anddioleoyl analogues and dihexadecyl, dilauryl, dimyristoyl anddipalmitoyl (Supelco and Calbiochem).

Monoglycerides are available from Sigma Chemical Co.(1-monopalmitoyl-(rac)-glycerol, monopalmitin;1-monocaprylol-(rac)-glycerol, monocaprylin; 1-monoleoyl-(rac)-glycerol(C18:1, cis-9), monoolein; 1-monostearyl-(rac)-glycerol, monostearin).

OTHER CONSTITUENTS

It is possible to add other constituents to the microcrystal to increaseits stability or modify its rate of release. For example,pharmacologically-acceptable oils can be added at low weightconcentration to facilitate contact between the microcrystal and thephospholipid or glycerol lipid coating. It is necessary that the type ofoil and its weight concentration be chosen such that the crystallinedrug not be dissolved by the oil and that the coating by themembrane-forming lipid not be disrupted. These relationships can bedetermined empirically. Useful oils include, but are not limited to,vitamin E, isopropyl myristate, benzyl benzoate, oleyl, alcohol, mineraloil, squalene and vegetable oil. Example 6 gives evidence thatincorporation of vitamin E in a lecithin-coated microcrystal preparationof erythromycin decreases the rate of dissolution of the drug, therebyreducing tissue irritation by the drug.

It is also possible to "precoat" the microcrystals byphospholipid-compatible, non-antigenic molecules which are solid at 37°C. Examples include paraffin, tristearin, ethyl oleate, cetostearylalcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol andpetrolatum. For example, these materials can be incorporated into theprimary coating by sonication or shear at temperatures above theirmelting points. Stabilization can be achieved by adding lecithin duringthe process as temperature is allowed to return to the solidificationpoint of these materials. It is desirable to use low weightconcentrations (≦10%) such that the payload is not degraded, the rate ofdissolution of the drug is not unduly impeded. Also, biodegradabilitymay impose a further limitation. Example 13 describes thelecithin-compatibility of paraffin in micro-particulate form.

SUSPENDING MEDIUM

In the final preparation, the continuous phase is generally water,buffered to a physiologically-acceptable pH and containing aniso-osmotic concentration of sodium chloride or glucose. In certainapplications involving intra-muscular injection of large volumes ofmicrocrystals at high concentration, it is useful to increase theosmolarity of the medium (e.g. glucose concentration) to facilitate thespreading of the material in the muscle. As noted above, this can retardthe process of compaction after intra-muscular injection. Wherepermissible, viscosity-increasing agents such as carboxycellulose can beuseful to alter the pharmacokinetics following intra-muscular injectionand to decrease the rate of sedimentation of the microcrystals uponstorage.

In certain applications it is useful to substitute a polar solvent forwater, as in Example 7 where albendazole sulfoxide did not showsufficient long-term stability in the presence of water. (Also seeExample 5.) Examples of non-aqueous polar solvents which can be usedinclude, but are not limited to the following: glycerin (water-miscibleliquid with a dielectric constant of 42.5) and propylene glycol(water-miscible liquid with a dielectric constant of 32). The coatedmicrocrystals can be made in these media, or can be allowed to sedimentinto these media. The primary requirement is that a substantial portionof the phospholipid or coating material be in membranous form in thissolvent. An equally important requirement is that the crystallinematerial not be sufficiently soluble in the solvent that it willrecrystallize.

PRESERVATIVES

Oil-soluble preservatives can be added in process during the primary orcoating phase. These include, but are not limited to, benzalkoniumchloride, propylparabum, butylparaben, and chlorobutanol. There are alsonumerous water- and oil-soluble agents which can be added to thefinished product as preservatives, including, benzyl alcohol, phenol,sodium benzoate, EDTA, etc.

OPTIONAL LYOPHILIZATION AND RECONSTITUTION

Aqueous microcrystal preparations can be lyophilized to give a dryproduct which can be reconstituted with water (Example 6). This isparticularly useful for a drug which does not have long-term stabilityin an aqueous environment. It is also possible to conduct the sonicationor shear process in a volatile organic solvent in which the crystallinedrug is not substantially soluble, and to prepare dry coatedmicrocrystals by solvent evaporation (Example 8). These procedures givemicrocrystals surrounded by layers of phospholipid in the anhydrousstate. Such forms are suitable for oral administration or forreconstitution with water and injection.

DESIGN OF THE FINAL PRODUCT

One skilled in the art following the instructions provided herein willhave no difficulty in empirically determining the:

Most convenient method of preparation:

Sonication vs. high pressure and shear vs. methods involving organicsolvents vs. impact in air vs. in-flight crystallization vs. solventdilution

Most advantageous form of the drug:

Crystal of neutral drug vs. crystal of charged drug vs. more complexsolid forms of the drug

Optimal membrane forming lipid:

Based on reactivity and stability of membranes, blood and tissuecompatibility and price

Optimal conditions for manufacture, including:

Input drug and phospholipid ratio; incorporation of small amounts ofoils or waxes as modifying agents; duration of sonication, shear, etc;use of sedimentation as a means of size selection; addition of moreperipheral lipid in unilamellar or multi-lamellar form; addition ofosmotic or viscosity-affecting agents.

Optimal particle size:

Which can be controlled to a certain extent by the power supplied, theduration of processing, the drug and phospholipid concentrations,

And which can be selected between 50 nm and 10 um by sedimentationvelocity

Optimal compositions to achieve the desired shelf life andpharmacokinetics:

Including study of the effect of the above factors on thepharmacokinetics after injection. Particularly important are theparticle size, primary and secondary phospholipid content, and additivesto avoid compaction after injection into a tissue.

Most advantageous mode of administration:

Including injection (IV, IA, IM, etc.), oral, topical administration,inhalation, etc.

WEIGHTS AND MEASURES

All parts and percentages reported herein are by weight (w/w) orweight/volume (w/v) percentage, in which the weight or volume in thedenominator represents the total weight or volume of the system.Concentrations of water soluble constituents in aqueous solution (e.g.glucose) are given in millimolar concentration (mM=millimoles per liter)referred to the volume of water in the system. All temperatures arereported in degrees Celsius. Diameters or dimensions are given inmillimeters (mm=10⁻³ meters), micrometers (um=10⁻⁶ meters), nanometers(nm=10⁻⁹ meters) or Angstrom units (=0.1 nm). The compositions of theinvention can comprise, consist essentially of or consist of thematerials set forth and the process or method can comprise, consistessentially of or consist of the steps set forth with such materials.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Lecithin-coated microcrystals of oxytetracycline (OTC) were prepared bysonication in the following manner:

Into a 150 ml glass beaker, 4.4 gm oxytetracycline dihydrate (Sigma,0-5750) and 16.0 gm egg lecithin (L-alpha-phosphatidylcholine from egg,Type XV-E, Sigma, P-9671) were added coarsely mixed using a glassstirring rod. Next, an aqueous solution of 300 mM glucose, 10 mM trisadjusted to pH 7.4, was added to give a final volume of 80 ml. Thebeaker was jacketed with a larger beaker filled with water which waskept in motion by means of a magnetic stirring bar. This, and occasionalinterruptions of the sonication, allowed for dissipation of heatproduced by sonication process. The 1.0 cm diameter probe of a Sonifier®Cell Disrupter, Model W185D (Heat System and Ultrasonics, Plainview,N.Y.) was immersed in the liquid and mixture was sonicated for a totalof 60 min at power stage 10 (nominally 100-150 watts). The temperatureof the mixture was not allowed to exceed 60° C. During the sonication,the preparation was titrated with 1.2M HCl to achieve a final pH of 5.0.Sonication resulted in an opaque yellowish-beige suspension. Next, thepreparation was covered and allowed to settle for 24 hrs. The bottom 11ml contained a visible precipitate of OTC at a concentration of 40%(w/v). The supernatant contained phospholipid vesicles. Bottom 22 mlwere collected and the precipitate was resuspended with gentle shaking.It contained lecithin-coated microcrystals of OTC. It behaved as asomewhat viscous but syringable liquid.

Fluorimetric analysis and high pressure liquid chromatographic (HPLC)showed that the top phase contained very little oxytetracycline. Thebottom phase sampled as the bottom 22 ml contained ≦98% of the addedoxytetracycline. It was 20% (w/v) in OTC and 20% (w/v) lecithin.Aliquots were taken from both phases and were diluted into OTC-saturatedbuffer and were analyzed for diameter (±SD) using a Coulter N4-MDSubmicron Particle Analyzer. The top phase was analyzed for particlesize was found to have diameters of 30.5±8 nm (77%) and <3,000 nmcorresponding to unilamellar and multilamellar phospholipid vesicles,respectively. The top phase was discarded. The lecithin-coated OTCmicrocrystal fraction had the following weight-averaged particle sizedistribution: 980±460 (SD) nm, 59%; 2,880±400 (SD) nm, 41%. Analysis ofthe preparation by electron microscopy using negative stainingcorroborated the above findings. Several preparations were made asdescribed above and were filled into rubber-stoppered glass ampules andglass bottles. With storage over a period of weeks some settling wasobserved, but the preparation could be rendered homogeneous with threeinversions. The preparation retained its properties, including sizedistribution, OTC concentration, chemical integrity and syringability(20 gauge or narrower) for over 9 months.

The importance of the lecithin coating was demonstrated as follows: 4.4gm OTC and 75.6 ml glucose solution were sonicated for 60 min asdescribed above, but in absence of lecithin. A coarse suspension wasobtained with the following characteristics: (a) Immediate sampling and1,000-fold dilution into OTC-saturated water gave particles visible tothe naked eye. Analysis by the Coulter Submicron Particle Analyzerreported that the particles were "out of range" (>3 um). The analysisdid not reveal any particles with diameters <3 um. (b) Within 10 minutesafter sonication, all of the OTC had settled to the bottom. The bottomphase was not free-flowing and was not syringable. Within 1 hr aftersettling, it became a solid mass which could not be resuspended withshaking. Similarly, it was impossible to stabilize this preparation byadding pre-formed phospholipid vesicles to the sonicated OTC immediatelyafter cessation of sonication. Thus sonication with lecithin (or othermembrane-forming lipid) is shown to be a critical step in the method ofpreparation.

EXAMPLE 2

The preparation of Example 1 was repeated with the followingalterations: The preparation was scaled to a total volume of 5.0 ml, themicrotip sonicator probe was used and 0.15 mg Nile Red was added at thesame time as the lipid. This dye binds to phospholipids and serves as afluorescent marker for the lecithin. A drop of the 20% (w/v) preparationwas spread on a glass slide and observed with a fluorescent microscope(Leitz Wetzlar Dialux 20) at high power. FIG. 2 is a black and whitedrawing of a typical field. White indicates high fluorescence. Withultra-violet excitation, OTC particles were observed by their intenseyellow-green fluorescence. The upper panel of FIG. 2 shows depicts thefluorescent image of OTC. Discrete particles with diameters ranging fromapprox. 0.2 um and ca. 3 um were observed. The majority of the particleswere ≦1 um diameter (number average). The particles had clear boundries,but were surrounded by diffuse yellow-green halo's. The identical fieldwas observed with near ultra-violet excitation to give the Nile Redimage associated with the lecithin in the preparation (bottom panel).The Nile Red image shows the lecithin to be surrounding the OTCparticles. A halo red fluorescence surrounded the particles, extending0.3 um to 3 um beyond the boundry of the OTC microcrystal. Empty spacesdevoid of both OTC and lecithin, could be readily discerned around allparticles situated near the edge of the smear. Occasionallyconfigurations were observed suggesting that two OTC particles weresharing a single phospholipid halo.

Brownian Motion was observed in the sample. The larger (>1 um diameter)particles which settled on the glass slide showed no Brownian Motion, orhighly restricted motion. Particles of ≦1 um diameter showed BrownianMotion, moving between the larger particles. Observations in regions oflow concentration showed that no particle could move a distance greaterthan approx. 1/4 its diameter without its halo experiencing acorresponding movement. Furthermore, direct collisions of the particleswere never observed. These observations explain the remarkable stabilityof the microcrystal suspensions of my invention: The lecithin coats themicrocrystal, supplying both a hydrophobic surface for contact with thecrystalline surface and a hydrophilic surface for contact with water.The coated surface is enveloped by numerous layers of lecithin inmembrane form, as revealed by the Nile Red staining. The stability ofthe envelopment is shown by the fact that the microcrystal alwaysremains within its lecithin halo, as revealed by their respectivefluorescence signals. The outer lecithin layers guarantee that thecoated microcrystals do not approach closely enough to fuse.

The dissolution behavior of the lecithin-coated microcrystals wasobserved under the fluorescent microscope by adding a large quantity ofdistilled water. The cover slip was raised and a drop of water wasbalanced next to it, making contact with the smear. The cover slip wasfloated, and the behavior of the preparation was observed. The smallerparticles (approx. 0.2-1.0 um) moved with the water flow. The Nile Redfluorescent halo moved together with the fluorescent OTC particle, andthe brightness of the two fluorescent signals initially remained inconstant proportion. As the particle moved into the distilled water, theOTC signal dimmed, suggesting that the microcrystal was dissolving. Onlya small fraction of the Nile Red image and intensity was lost,suggesting that the lecithin coating was a persisting structure. Thedissolution behavior of the larger particles which were generally morefirmly attached to the slide was somewhat different. The streamingcaused them to shed a large portion of their lipid. The largermicrocrystals were then observed to crack, splitting off shards each ofwhich carried away a portion of the Nile Red halo with it.

Dissociation behavior was also studied using the Coulter N4-MD SubmicronParticle Sizer. As stated in Example 1, when the preparation is diluted1,000× into isotonic glucose buffer saturated with OTC, the preparationis stable and particles sizes of 980±460 nm and 2,880±400 nm wereobserved. When the dilution was made 1,000× into distilled water, rapidalteration of the preparation was observed. Dissolution was expectedsince the final OTC concentration becomes 0.2 mg/ml, which is lower thanthe aqueous solubility of the drug (about 1 mg/ml). Dissolution wasobserved, but it was accompanied by the formation of some particles withdiameters greater than 3 um.

EXAMPLE 3

A Nile Red "doped" lecithin-coated microcrystal preparation ofoxytetracycline was made and the following fractionation experiment wascarried out to delineate the relationship between the primary andsecondary phospholipid coatings for the larger-diameter (1.0-1.9 um)coated microcrystals.

Oxytetracycline (2.0 gm), lecithin (8.0 gm) and Nile Red (1.5 mg) wereadded to 40 ml of isotonic glucose and sonicated for 30 min. Thepreparation was allowed to concentrate to 20% (w/v) OTC by sedimentationovernight. The volume of this preparation was 10 ml. Small aliquots weretaken for fluorimetric assay of Nile Red concentration, phospholipidanalysis (by ammonium ferrothiocyanate extraction) and for observationunder a fluorescence microscope. Then the preparation was centrifugedwith a clinical centrifuge at medium speed for 15 min. This resulted ina visible precipitate of 2.0 ml volume. The top phase was separated, anda aliquots were analyzed (1st supernate). The bottom phase wasresuspended to a final volume of 10 ml by addition of isotonic glucose.Aliquots of this were analyzed (1st wash). This was repeated to give atotal of 5 washings. The procedure removed the small (0.1-0.3 um)diameter coated microcrystals and loosely attached phospholipidvesicles. This allowed the large (1-3 um diameter) coated microcrystalsto be isolated and enabled their phospholipid/drug ratio to be assessed.

                                      TABLE 1                                     __________________________________________________________________________    Step [OTC]                                                                             [Nile Red]                                                                          [Lecithin]                                                                             Microscopic Observations                              __________________________________________________________________________    Prep.                                                                              19.55%                                                                             100 units                                                                          127 ± 7                                                                         mg/ml                                                                             Observed OTC crystals 0.1-1.5 um diameter. All                                crystals had bright Nile Red halo                                             with outer diameter ca. 2 × that of                                     crystals. Small crystals and their halos                                      showed Brownian Movement between larger crystals                              and their halos. The                                                          latter were largely stationary.                       1st wash                                                                           19.55%                                                                            18.7 units                                                                          20 ± 3                                                                          mg/ml                                                                             Observed OTC crystals 0.1-1.9 um diameter.                                    Smaller crystals (0.1-0.3                                                     um) were in lesser abundance. A substantial                                   fraction was lost to                                                          supernate of first wash.  Brownian Movement was                               as described above. Nile                                                      Red halos were reduced to ca. 1.5 × the                                 crystal diameter and were dimmer.                     2nd wash                                                                           18.89%                                                                             2.6 units                                                                          247 ± 75                                                                        ug/ml                                                                             Sizes, composition and movement were similar to                               those observed in 1st                                                         wash, but the Nile Red intensity was much lower.      3rd wash                                                                           18.89%                                                                             0.7 units                                                                          99 ± 10                                                                         ug/ml                                                                             Sizes, composition and movement were similar to                               2nd wash, but the Nile                                                        Red intensity was still lower.                        4th wash                                                                           18.40%                                                                             0.3 units                                                                          53 ± 53                                                                         ug/ml                                                                             Sizes, composition and movement were similar to                               3rd wash, but Nile Red                                                        intensity was very faint on large crystals, and                               not visible on small crystals.                        5th wash                                                                           17.07%                                                                             0.4 units                                                                          160 ± 50                                                                        ug/ml                                                                             Same as 4th wash, but crystals were grouped in                                clusters of 5-10.                                     __________________________________________________________________________

The observation show that as the larger crystals are repeatedly washedthey lose the greatest fraction of their associated lecithin. The amountof associated lipid stabilizes between at the 3rd-5th wash at 108±80ug/ml or approx. 0.4% of its input value (Nile Red). The thickness ofthis coating can be estimated from the volume relationships,approximating the density of the OTC and the lecithin as equal (ca. 1.4gm/cm³). From the Nile Red data the thickness of the layer 15 Angstromunits. This is close to what is expected from a monolayer of extendedlecithin molecules. From the phospholipid analysis the estimate is lower(ca. 3 Angstrom units) but the experimental uncertainty is large and theextraction efficiency for the 3rd to 5th samples may have beenconsiderably less than one. The above procedure may underestimate thethickness of the enveloping layer if the latter were stripped off by theforces of centrifugation. For the small (0.1-0.3 um diameter)microcrystals, the Nile Red halo is observed to be tightly associatedwith the microcrystal while undergoing Brownian Motion. This suggeststhat its enveloping layer is quite stable.

EXAMPLE 4

The pharmacokinetics of lecithin-coated oxytetracycline microcrystalswere determined in laboratory rats. The preparation was made essentiallyas described in Example 1. It contained 24% (w/v) OTC and 20% lecithin.Samples (0.1 ml) were administered by deep intramuscular injections intothe hindlegs of laboratory rats. Injections were made (distal toproximal) into the gastrocnemius. Serving as a positive control were 0.1ml injections of a commercial sample of IM-injectable OTC (Liquamycin®200, Pfizer), consisting of 200 mg/ml OTC base as amphoteric OTC, 40%(w/v) 2-pyrrolidone, 5.0% povidone (w/v), 1.8% (w/v) magnesium oxide,0.2% (w/v) sodium formaldehyde sulfoxylate and monoethanolamine and/orHCl as required to adjust the pH. At designated times central arterialblood was taken, the animals were sacrificed and the injected muscleswere dissected out and examined grossly and under ultra-violet light foroxytetracycline fluorescence. The blood samples and muscles wereextracted with ethanol and the oxytetracycline concentration wasdetermined fluorimetrically.

FIG. 3 shows that the oxytetracycline is released slowly from the musclewhen injected in the lecithin-coated microcrystal form, with approx. 20%of the injected dose remaining in the muscle after 7 days. The releaseis substantially slower than the commercial pyrrolidone solution. FIG. 4shows that blood levels from 4 ug/ml to 1.5 ug/ml are sustained over a7-day period. This can be compared with the commercial solution forwhich the blood levels drop to 0.5 ug/ml or less within 3 days.

EXAMPLE 5

A large number of preparations of lecithin-coated oxytetracyclinemicrocrystals were formulated at concentrations between 20% (w/v) and44% (w/v), as described above. The present Example shows how thesecondary coating (peripheral vesicles) can be added after the initialsonication step, and how hypertonic glucose and viscosity-increasingagents can be included within its entrapped aqueous volume. Twenty gm ofegg lecithin and 5 gm OTC dihydrate were placed in a beaker and anaqueous solution of 12.5% (W/v) glucose, and 10 mM tris buffer, pH 7.4was added to obtain a final volume of 100 ml. Sonication and pHadjustment were done as in Example 1. Four separate batches were pooledand stored overnight under refrigeration in at screw cap container.After the preparation had settled, the top 87.5% (350 ml) was drawn offleaving lecithin-coated OTC microcrystals and a small portion of the topphase (phospholipid) in suspension. Analysis showed that the lower phasecontained 40% (w/v) OTC. (Preparations of OTC could be concentrated to44% (w/v) by sedimentation.)

Peripheral phospholipid vesicles were prepared separately and admixedwith the concentrated lecithin-coated microcrystals. Five gm egglecithin and 0.1 gm propylparaben (preservative) were added to 45 ml ofan aqueous solution of 12.5% glucose and 5% carboxymethylcellulose andthe mixture was sonicated at power level 8 for 15 min. This resulted ina thick but syringable suspension of lecithin vesicles entrapping thecarboxymethylcellulose and hypertonic glucose.

To complete the formulation, 33 ml of the 40% OTC lecithin-coatedmicrocrystal preparation were mixed with 33 ml of the above peripherallipid preparation. The mixture was stored in sealed ampules. The finalconcentrations were 20% (w/v) OTC, 15% (w/v) lecithin, 0.1% (w/v)propylparaben, with the aqueous phase consisting of 12.5% (w/v) glucoseand 10 mM tris, pH 5.0.

Experimentation with IM injection in rat showed that preparations madein hypertonic glucose or carboxymethylcellulose, or admixed withperipheral lipid sonicated in the presence of these agents had fasterremoval of OTC from the injection site than isotonic controls.

The above preparation (denoted Formulation F) was injectedintramuscularly into three approx. 300 lb calves at a dose of 9 mgOTC/kg body weight. FIG. 5 shows the average (±SE) blood OTCconcentrations as a function of time after injection. The bloodconcentration vs. time curve is flat, maintaining concentrations between0.5 ug/ml and 1.0 ug/ml in the time range between 2 and 120 hrs (5days). Sustained release of this type can be useful, because withsuitable high dosing, the animal can receive therapeutic concentrationsover a period of 5+ days, without the need for repeated injection.

Results similar to those of FIG. 5 were obtained with the followingcompositions:

                  TABLE 2                                                         ______________________________________                                                       Treatment of                                                   [OTC] [Lecithin]                                                                             peripheral lecitin                                                                         [glucose]                                                                            Polar phase                                ______________________________________                                        20%   30%      homogenized  21%    water                                      20%   30%      homogenized  12%    water                                      20%    5%      sonicated     0%    45% propylene                                                                 glycol;                                                                       30% water                                  ______________________________________                                    

All of these samples showed good syringability and physical stability.There were subtle differences in their pharmacokinetics. They gave nopain upon injection or swelling of the injection site. The lack of painon injection is a particular advantage over commercial solutions.

EXAMPLE 6

Erythromycin, an antibiotic with poor aqueous solubility, was alsoformulated as lecithin-coated microdroplets in a manner similar to thatof Example 4. The aqueous solubility of erythromycin is higher thanoxytetracycline at neutral pH (2 mg/ml vs 1.1 mg/ml). Erythromycin isknown to be irritating to tissue at high concentration. This wasverified by my experimentation, in which a slurry of erythromycincrystals (20% w/v) suspended in propylene glycol was injectedintra-muscularly (rat). This resulted in extensive pain on injection anddamage, such that the rat had to be sacrificed immediately. Thefollowing example illustrates how use of lecithin coating reduces theirritation intrinsic to the drug (also see Example 6) and how theirritation can be further reduced by incorporation of a water-insolublepharmacologically-acceptable oil in the lecithin-coated microcrystal.Table 3 presents results for two compositions prepared by sonication andtested by IM injection in rats.

                  TABLE 3                                                         ______________________________________                                                                 Obs. with 0.1 ml IM                                  Composition Characteristics                                                                            injection                                            ______________________________________                                        20% Erythromycin                                                                          0.7 um particles;                                                                          Difficulty in walking                                15% Lecithin                                                                              syringable; settles                                                                        apparent pain; sacrified                             Remainder:  in 2 days, resusp.                                                                         at 4 hours; obs. drug                                5.4% glucose                                                                              with inversion                                                                             deposit and extensive                                                         hematoma                                             20% Erythromycin                                                                          0.7 um particles;                                                                          No abnormal behavior                                 15% Lecithin                                                                              syringable; settles                                                                        or pain on inj.; sacrificed                          5% Vitamin E                                                                              in 2 days, resusp.                                                                         after 4 days; little or no                           Remainder:  with inversion                                                                             drug deposit; small                                  5.4% glucose                                                                              hematoma.                                                         ______________________________________                                    

The data show that when vitamin E is added, the drug-induced damage isreduced to acceptable levels. The extra protective effect of vitamin Eis most likely due to its ability to insert between erythromycinmicrocrystal and the lecithin coating, creating an additional buffer andbarrier towards erythromycin dissolution. This serves as an example inwhich the lipid coating is modified to reduce the availability of theincorporated drug.

My experimentation with erythromycin formulations has also definedconditions under which lyophilized forms of phospholipid-coatedmicrocrystals are useful. A preparation of 20% erythromycin, 15%lecithin was frozen and placed in a lyophilization apparatus to yield apowder. Upon mixing and swirling with 12% (w/v) glucose it produced asuspension which was syringable and physically stable. Particle sizinggave an average diameter of 0.7 um, identical to the originalpreparation.

Further experimentation showed that an equivalent lyophilized productcan be produced without using water. Sonication was performed in thefluorocarbon Freon TF (trichlorotrifluroethane): Into a sonicationvessel, 2 gm erythromycin were mixed with 0.5 gm egg lecithin using aglass stirring rod. Next, 7.5 ml Freon TX was added to the mixture. Themixture was then sonicated at power level 5 for 30 min using themicrotip to yield a homogeneous suspension (vessel water jacketed;sonicator cycled on and off). The suspension was placed in a 250 mlErlemeyer flask and nitrogen gas was blown over the sample. The flaskwas rotated to distribute the drying material evenly on the bottom. Theremaining solid material was dried in a Labconco Bench Top Lyophilizer(Model 75034) overnight. The resulting material was yellowish pelletsthat resuspended with moderate shaking in a diluent consisting of apresonicated aqueous suspension containing 20% lecithin and 5.4%glucose. The resuspended erythromycin was a syringable, physicallystable suspension of 20% (w/v) erythromycin in the form of 0.7 umdiameter particles (Coulter N4 Particle Sizer).

Additional experimentation showed that this procedure requiressonication in a solvent such as Freon, in which the solubility of theantibiotic is minimal. The above procedure was repeated, with thesonication taking place in chloroform or ethanol, in which theerythromycin is soluble. In both cases a powder was obtained which didnot properly resuspend when added to an aqueous medium. This defines arequirement for the drug to remain almost exclusively in the crystallinestate during dispersion and evaporation processes.

EXAMPLE 7

The lecithin-coated microcrystal form has also proven useful for theadministration of anthelmintic drugs. For these applications, it isdesirable for the drug to be absorbed from the IM injection site intothe system within 2 days. A preparation of 10% (w/v) albendazole (8%)lecithin-coated microcrystals was found to give 470 nm diameterparticles (Coulter N4 Particle Sizer), to be syringable, to be grosslystable for 60 weeks. Albendazole preparations could be concentrated bysedimentation to 20% (w/v). Upon IM injection of 25 ul into the hindlegs of laboratory rats resulted in the following pharmacokineticbehavior:

                  TABLE 4                                                         ______________________________________                                        Time,                                                                         post-inj.                                                                              Drug in inj. site                                                                             [Drug] in blood                                      ______________________________________                                         4 hr.   2.0 ± 0.7 mg (80%)                                                                         112.5 ± 11.0                                                                          ug/ml                                      8 hr.   2.4 ± 0.5 mg (97%)                                                                         62.5 ± 7.8                                                                            ug/ml                                     24 hr.   1.7 ± 0.5 mg (69%)                                                                         <20        ug/ml                                     ______________________________________                                    

Gross observations of the muscles prior to extraction showed drugdeposits with no observable damage to the surrounding tissue.

Injections of 10-20 ml of this preparation intra-muscularly orsub-cutaneously into sheep were well-tolerated. Necropsy at 7 daysrevealed deposits representing a major fraction of the drug, with littleirritation of the surrounding tissue. The slow absorption of the druginto the circulation was probably the result of large volume injectedand the low aqueous solubility of albendazole (<0.1 mg/ml). This limitedthe therapeutic usefulness of the lecithin-coated microcrystal injectionform for albendazole, but portends usefulness for other drugs of lowwater solubility for which slow release over periods of weeks isdesired.

The above experimentation was repeated with albendazole sulfoxide, amore water-soluble analogue of the drug. Faster rates of absorption intothe blood were obtained. Solubility data for albendazole sulfoxide are:0.42 mg/ml in water, 17.7 mg/ml in propylene glycol and 179.0 mg/ml inethanol. Preparations were made with the following compositions: (A) 20%albendazole sulfoxide, 20% egg lecithin, in 5.4% buffered aqueousisotonic glucose; (B) 20% albendazole sulfoxide, 5% lecithin, inpropylene glycol. Both preparations normally settled to albendazolesulfoxide concentrations of 20% (w/v). Samples (0.1 ml) of bothpreparations were injected IM in rats (n=6) and the muscles wereexamined at necropsy, 2 days post-injection. Both preparations showedsmall deposits of drug and no irritation of the surrounding tissue.Propylene glycol, which has two OH groups and a dielectric constant of32, is an example of a polar organic compound which can be substitutedfor water. In present case, this proved advantageous to the long-termchemical stability of the drug and hence the shelf-life of thepreparation.

Experiments with calves showed that a microcrystal preparation of 15%(w/v) albendazole sulfoxide, 3.75% (w/v) lecithin in propylene glycolwas particularly useful. Intramuscular injection of ca. 6 ml of thispreparation into calf (dose=6 mg/kg) resulted in no pain, and barelynoticable swelling at 24 hrs. No drug residue and only slight musclediscoloration were observed upon necropsy after 7 days. Similar resultswere obtained with 15% (w/v) microcrystalline albendazole sulfoxide plus5% egg lecithin suspended in a 70/30 mixture of propylene glycol andwater. Both of these preparations satisfy current criteria for theusefulness of the preparation in veterinary medicine. Control studies ofthese preparation without added lecithin showed the drug to beintrinsically irritating, as verified by the animal's behavior, grossobservations and histology. This shows that the lecithin-coating helpsto reduce the local reaction to this drug.

EXAMPLE 8

Nitroscanate is a water-insoluble anthelmintic compound. An IV- or IMinjectable form would be desirable. The compound is not chemicallystable in water or in the presence of high relative humidity. It is alsochemically reactive with amines. Lecithin-coated microcrystals were madeby sonication in Freon as described in Example 6 and were stored inpowder form. Reconstitution with aqueous vehicle yields an injectableproduct. Reconstitution of this product with 5.7 volumes of 20% (w/v)sonicated lecithin containing 0.1% propylparaben in 12.5% glucoseyielded a 10% (w/v) suspension of nitroscanate microcrystals. Thesuspension was syringable and stable for several hours. The particlesize was approx. 500 nm, as estimated by microscopic evaluation.

EXAMPLE 9

This example shows how the phospholipid-coated microcrystal can be usedas a delivery system for non-steroidal anti-inflammatory drugs in thecontrol of inflammation. The preparation can be injected to create anintra-muscular depot, or can be injected into the tissue to beprotected. Indomethacin was taken as an example. The molecule is acarboxylic acid with a pKa of 4.5, but its aqueous solubility at pH 7.0is only 0.376 mg/ml. To produce a 3% (w/v) solution it is necessary toraise the pH to 9.6. Although its water solubility is poor, themolecule, in the diluted state, shows only moderate oil/water partitioncoefficients: 55/1, olive oil/water; 85/1, pentanol/water.

A lecithin-coated indomethacin microcrystal preparation was made by thefollowing procedure: Indomethacin (500 mg) was mixed with egg lecithin(2.0 gm) with a glass stirring rod, and an aqueous solution of 300 mMglucose, 10 mM tris (pH 7.4) was added to a final volume of 10 ml. Themixture was sonicated for a total of 30 min at power level 6 using themicrotip, with water jacketing and cycling as described previously. Thisresulted in a homogeneous suspension of coated microcrystals. Thepreparation was allowed to concentrate by sedimentation to give a finalcomposition of 20% (w/v) indomethacin and 20% (w/v) lecithin. (Thehighest concentration achievable was 25% (w/v) indomethacin.) An averageparticle size (Coulter N-4 Particle Sizer) of 100 nm was determined.Long-term stability was good. There was further settling but thepreparation could be resuspended with three inversions.

The above preparation was tested in rat as an intra-muscular depot foranti-inflammatory activity using the Carrageenan-induced paw edemamodel. The dose of was 5 mg indomethacin given by IM injection into therear leg of 0.025 ml to the preparation or 0.0325 ml of a 15.4% solutionat pH 10.5. Efficacy of inhibition of the paw edema was evaluated afterCarrageenan challenges at 1, 24, 48 and 72 hr, post-injection. Therewere 3 rats per time and treatment group. The animals were sacrificedafter testing and gross observation of the muscles were made atnecropsy. Injections of the microcrystal preparation were not painful asevidenced by lack of appreciable vocalization or retraction of legduring injection and by normal use of the injected leg in the 3-hrperiod after injection (12 of 12 animals). In contrast, injection of thealkaline solution resulted in vocalization, retraction and limping onthe injected leg (12 of 12 animals). FIG. 6 shows the time course of theaverage percent (±SD) protection against paw edema (opposite leg) afterCarrageenan challenge at 72 hr for the two forms. The figure shows thatthe microcrystal preparation gives 89% while the alkaline solution givesonly 38% protection. The rats challenged at 72 hrs were sacrificed andthe injected muscles were examined at necropsy. The indomethacinmicrocrystal-injected muscles appeared normal (3 of 3 animals). Incontrast, two of the three muscles injected with the alkaline solutionshowed damaged areas of ca. 3× 6×1 mm dimension, with markeddiscoloration.

The above demonstrates the ability of the microcrystal formulation tointroduce high concentrations of drug into the tissue with minimalirritation. This indicates utility as a vehicle for anti-inflammatorydrugs, both in IM depot injections and in injection into the inflammedtissue or space (e.g. synovial fluid).

EXAMPLE 10

This example shows that phospholipid-coated microcrystals are capable ofrapidly releasing their contents when injected intravenously. Thewater-insoluble steroid anesthetic alfaxalone is delivered by thismechanism, becoming available to the brain within 10 sec of itsintra-venous injection.

A lecithin-coated alfaxalone microcrystal preparation was made bycosonication of the two constitutents in an aqueous solution 300 mMglucose, 10 mM tris buffer, pH 7.4 to give a preparation with 2% (w/v)alfaxalone, 2% (w/v) egg lecithin. The microcrystals were 548±75 nmdiameter (Coulter N4 Sub-Micron Particle Sizer). The preparation wasstable for upwards of 4 months. It separated to give a free-flowingsediment which mixed with inversion, and was completely resuspended withshaking.

The following data show that the preparation gives rapid generalanesthesia with intra-venous injection: The above preparation (0.11-0.25ml) was injected intravenously (tail vein) into 200-250 gm female rats(Harlen S-D) to give a doses of 10, 15 or 20 mg/kg. A dose of 10 mg/Kgrendered 6 of 10 rats unconscious. This dose thus approximates the EC₅₀.Doses of 15 mg/Kg and 20 mg/Kg rendered all of the animals unconscious(3/3 and 10/10, respectively). The animals were rendered unconsciouswithin 10 sec of commencement of injection. Anesthesia was as fast asthe injection which itself required 15 sec. Four types of qualitativeand quantitative data were recorded as a function of dosing level: (a)Characteristic spontaneous behavioral changes with emergence fromanesthesia, (b) threshold for vocalization with electrical stimulationand (c) surgical anesthesia tested by abdominal incision.

(a) The first indication that the process of emergence had begun was theonset of periodic spasms. For dosing at 20 mg/Kg these occurred at30.6±16.8 min (±SD, n±10). This was followed by arousal and strugglingto right which occurred at 50.6±22.0 min. righting at 57.9±22.4 min. Theanimals regained normal responses and spontaneous behavior at 84.0±25.5min.

(b) The threshold for vocalization to electrical stimulation viaintra-dermal electrodes was tested (needle electrodes in the skin of theback, 3 mm apart; Grass S44 Stimulator and Stimulus Isolation Unit;square waves of 1.5 msec width at 50 Hz; duration of stimulus=2 sec;amplitude measured in mAmp). Vocalization thresholds>7.0 mAmp representvery high anesthesia; thresholds in the >2.0 mAmp range representintermediate level anesthesia. In unanesthetized human skin (intradermalinsertion in leg) a 7 mAmp stimulus causes intolerable pain and a 2.0mAmp stimulus causes sharp pain. At all three doses, all rats which wererendered unconscious showed immeasurably high thresholds forvocalization: No vocalization was obtained for a maximal 15 mAmpstimulus. FIG. 7 shows typical results for individual rats. Thefollowing summarizes the observations with the three dosage groups (3rats per group): Immeasurably high anesthesia lasted for at least 5 minfor all the animals rendered unconscious at the 10 and 15 mg/Kg doses,and for at least 15 min for all animals at the 20 mg/Kg dose. Theduration of high level anesthesia increased as a function of dose withtimes of 26.7±20.8 min for 10 mg/Kg, 35.0±20.0 min for 15 mg/Kg, and41.7±7.6 min for 20 mg/Kg. The corresponding times for intermediatelevel anesthesia were 35±15.0 min, 66.7±12.6 min and 63.3±15.3 min.Thresholds of all treated rats returned to baseline levels within twohours of injection.

(c) Surgical anesthesia was tested in a separate group of rats at timesapproaching the longest time at which the electrical threshold was >15mAmp. At 10 mg/Kg dose, none of three animals tested responded toincisions opening the abdominal cavity at t=15 min post-injection. At 20mg/Kg, none of the three animals tested responded to opening theabdominal cavity at t=45 min post-injection. (Immediately after thisdemonstration of surgical anesthesia, the animals were sacrificed by CO₂asphixiation.)

The above data show that lecithin-coated alfaxalone microcrystals candissolve and enter the brain and produce general anesthesia within 10sec of their intra-venous injection. The data also show that thepreparation has utility as an induction agent and as an injectablegeneral anesthetic.

EXAMPLE 11

In this example, the alfaxalone preparation of Example 10 ischaracterized with respect to structure and dissolution behavior. A 2%(w/v) alfaxalone preparation was made by sonicating the drug at 2% (w/v)together with 2% (w/v) egg lecithin and 0.05% Nile Red in a medium of300 mM glucose, 10 mM tris (pH 7.4) at high power for 20 min. As inExample 3, Nile Red, a fluorescent dye which has affinity for lipids andphospholipids, was used to visualize and track the lecithin in thepreparation. Analysis with the Coulter N4 Sub-Micron Particle Analyzerimmediately after dilution into an alfaxalone-saturated solution gave anaverage particle diameter of 0.52±0.03 um. Visualization of thepreparation on a slide using a Leitz Wetzlar Dialux 20 FluorescentMicroscope revealed particles of this size. The particles consisted ofcolorless birefringent crystals of spherical or rounded shape surroundedby an intense halo of red fluorescence with a diameter of approx. 1.2 to1.7 times that of the crystal. The fluorescence was intense near thecrystal surface and was progressively more diffuse as a function ofdistance from the crystal surface.

The proportion of primary vs secondary lecithin coating was determinedin a fractionation experiment of the type described in Example 3. Thepreparation was sedimented in a clinical (blood) centrifuge at mediumspeed for 15 min. The supernate was drawn off. Aliquots (10 ul) of theoriginal preparation and its supernate were added to a cuvettecontaining 2.5 ml ethanol and Nile Red fluorescence (Fl) was measured ina fluorometer. The precipitate was resuspended in the glucose/trismedium to reconstitute the original volume, small portions were removedfor analysis with the particle sizer and microscopic observation. Thissequence was repeated for a total of three times. Table 5 shows thebehavior of the particle size.

                  TABLE 5                                                         ______________________________________                                        Behavior of Alfaxalone Preparation with                                       Repeated Centrifugation and Resuspension                                             Nile Red Fluorescence (Fl)                                                                    Particle                                               Preparation                                                                            Fl. Total Fl. Supernate                                                                             Diameter                                       ______________________________________                                        Original 292.0     310.0       0.52 ± 0.03                                                                         um                                    1st Resusp.                                                                             24.0      13.5       2.4 ± 0.7                                                                           um                                    2nd Resusp.                                                                             9.5       5.0        >3.0     um                                    3rd Resusp.                                                                             6.5       6.5        >3.0     um                                    ______________________________________                                    

Microscopic observation of the 1st resuspension showed that themicrocrystals retained their Nile Red halos, but that the halos weremuch thinner and less intense. Also, the microcrystals were clumped inaggregates of ca. 2.5 um diameter. Thin layers of Nile Red were visiblebetween the microcrystals in the aggregate. Observation of the 2nd and3rd resuspensions revealed still larger aggregates (of diameter approx.8× that of particles of the original preparation). The aggregates showeda faint pink fluorescence.

The fluorescence data in Table 5 show that 98% of the lecithin in thepreparation was peripheral phospholipid (FIG. 1) which could bedissociated by washing. The 2.22% of the lecithin, as reported by NileRed fluorescence, is tightly associated with the alfaxalonemicrocrystals. This represents the primary coating. Assuming equaldensities of the drug and lecithin, one can readily calculate thatdistribution of this amount of lecithin on a 520 nm diametermicrocrystal would result in a layer 1.9 nm or 19 Angstroms thick. Thisis very close to the expected thickness of a monolayer of lecithin. Theexperiment also demonstrates the role of the peripheral lecithin inpreventing aggregation of the microcrystals. Its removal allows themicrocrystals to come into closer proximity and to be aggregated by theaction of long-range forces.

EXAMPLE 12

The following example shows how the phospholipid-coated microcrystal canbe used as a means of producing long-duration anesthesia of the skinwith a single injection. Cherney (U.S. Pat. No. 2,803,582, 1957)described how the water-soluble local anesthetic tetracaine can berendered water insoluble by formation of the hydroiodic acid (HI) salt.Goodman and Gillman's "The Pharmacological Basis of Therapeutics (7thEd., MacMillan Publishing Co., New York, 1985, p. 312) cite a study(Cherney, L. S. Anesth. Analg. 42:477-481, 1963) showing that crystalsof this salt can be sprinkled into surgical wounds to provide localanesthesia of 45 hr. duration. However, reference to the 1988 PDRindicates that hydroiodic acid salt of tetracaine is not commerciallyavailable for clinical use in the U.S. The present example shows how theutility of Cherney's invention can be increased by making itlecithin-coated microcrystals.

Insoluble tetracaine-H-I was prepared by adding potassium iodide (KI) toa saturated aqueous solution of tetracaine-H-Cl. The precipitate wasresuspended and washed several times with water and then dried. Forpreparation A, 10 gm of tetracaine-H-I and 1 gm egg lecithin were addedto a test tube, and 5.4% glucose, 10 mM tris, pH 7.0 was added to afinal volume of 10 ml. The material was sonicated (with temperaturecontrol) for a total of 20 min to yield a white suspension. This wasallowed to settle overnight, and the top half was discarded. PreparationB was made in a similar manner. Tetracaine-H-I (0.50 gm) and egglecithin (1.0 gm) were consonicated in 10 ml volume. The top 7.5 ml werediscarded and the bottom 2.5 ml were resuspended to give the finalpreparation. Both preparations were 20% (w/v) tetracaine-H-I and 10%(w/v) egg lecithin. Both preparations showed the tendency to sediment togive 30% (w/v) tetracaine-H-I. The long-term stability of bothpreparations was good.

Preparation A (0.1 ml) was injected intradermally in the skin of thebacks of rats raising a ca. 1.0 cm diameter wheal which was demarcatedwith a felt tip pen. The degree of anesthesia in the injected skin wasdetermined by the shock vocalization test using indwelling intra-dermalelectrodes positioned in the center of the injected area. The thresholdfor vocalization was immeasurable high (>15 mAmp) during the first threehours after injection. Four rats were tested during the time-interval22-25 hr. post-injection. This group had an average (±SD) vocalizationthreshold of 6.6±2.6 mAmp, indicating good anesthesia. Retesting of thisgroup at 41-44 hr. showed that the anesthesia had subsided. For three ofthe four animals the skin appeared normal. One animal showed an approx.2 mm diameter brownish spot in the middle of the injected skin. As acontrol, two animals were injected with 0.1 ml of tetracainehydrochloride solution. This resulted in high levels of anesthesia (>15mAmp) in the initial, but the anesthetic solution caused severe damageto the tissue with scabbing observed on the second day such thatmeasurement of anesthesia was neither practical nor meaningful. This wasverified by the results in two additional rats with two injections each.All four injected areas were completely brown and scabbed at 24 hrs andcraters were observed at 48 hrs.

The Inventor carried out self-experimentation with Preparation B. I madetwo intradermal injections of 0.15 ml of Preparation B into the skin ofmy calf, right leg, inside, at sites 12 cm and 22 cm below the knee. Theinjections raised weals approx. 1.1 cm in diameter. There was no pain oninjection. The weals subsided within ca. 30 sec. The injected sites weretested for pin prick and cold stimulus anesthesia for the next 24 hr.FIG. 8 shows pin prick anesthesia on a 5-point scale (4/4=fullinsensitivity, 0/4=full sensitivity, to the sharpness of the pin). Thefigure also shows observations with 2% and 5% tetracaine-H-Cl solutions.The lecithin-coated microcrystal preparation showed complete anesthesiafor 7-9 hrs after the injection, with return to 50% sensitivity at121/2-141/2 hrs, and complete reversal at 16-21 hrs. The injection didnot produce irritation. At 11 min or 11/2 hr, a slight erythrema wasobserved. The injected areas appeared and felt completely normal at 24hrs. The only reliable means of differentiation of injected anduninjected tissue was a greater sensitivity to vigorous rubbing 1-5 dayspost-injection.

As a control for the above, I injected myself with 0.15 ml volumes ofsolutions of 1%, 2% and 5% tetracaine-H-Cl. In attempt to make theconcentrated solutions less damaging, the pH was adjusted from 5.3 to6.5. Physiological tonicity was maintained by including glucose (4.3%,3.2% and 0%, respectively). FIG. 8 shows that these solutions producedfull anesthesia of not more than 2 hr. duration. The 1% and 2%tetracaine-H-Cl solutions produced only mild erthrema, with return tonormal color when the anesthesia subsided. The 5% tetracaine-H-Clsolution produced a bright red spot (7 mm diameter) in the center andhardness at 27 min. The presented anesthesia values were taken at itsperiphery. The site was sore after anesthesia had subsided. The red spotresolved into a scab at 7 days which persisted to 21 days. At 48 days(the time of this writing) the site has a 2 mm diameter scab surroundedby a 1 cm diameter circle of pinkish skin, sensitive to the touch andraised approx. 1 mm. This poor outcome with the 5% (w/v) tetracainesolution is in stark contrast to the excellent results obtained with the20% (w/v) tetracaine-H-I microcrystal.

The above data show that the use of the lecithin-coated microcrystalmethod in conjunction with the invention of Cherney allows theanesthetic to be injected at over 4 times higher concentration,producing safe, reversible anesthesia of 5 times longer duration.

EXAMPLE 13

This example shows that particles of waxy substances can be coated andstabilized with a layer of lecithin. These phospholipid-coatedmicroparticles can be made from phospholipid-compatible solid materialswhich melt between physiological temperature (37° C.) and 100° C. Thepresent example illustrates this using paraffin wax. Paraffin (3.35 gm)was melted in a water bath at 60° C. Egg lecithin (1.35 gm egg lecithin)was put in a beaker and an aqueous solution of 300 mM glucose, 10 mMtris (pH 7.0) was added a final volume of 47 ml and homogenized. Theliquid paraffin was added to the homogenized lecithin and the mixturewas sonicated for 30 min to obtain a milky uniform suspension. Thebeaker was covered and allowed to cool to room temperature. The resultwas a suspension of lecithin-coated submicron diameter paraffinparticles which was stable in excess of two weeks. Repetition of theabove in the absence of phospholipid resulted in precipitation of solidparaffin.

The above example shows that it is possible to make lecithin-coatedmicroparticles from a material with a melting point below the boilingpoint of water and above that of the intended temperature of use (37°C.). Pharmaceutically acceptable waxes and solids, of biological andsynthetic origin, include but not limited to hydrogenated castor oil,cetostearyl alcohol, cetyl alcohol, cetyl esters wax, myristyl alcohol,petrolatum, paraffin, and various waxes (emulsifying, microcrystalline,white, yellow, etc.). It is also possible to use these materials toprovide a waxy coating for the drug microcrystals, which is in turncoated with phospholipid. The waxy coating will further slow the rate ofrelease of the drug, thus prolonging its duration of action.

Lecithin-coated microparticles of paraffin, or alternatively tristearinas a biodegradable wax, will have very long lifetimes in injectedtissues. It is likely that they will be useful for the fixation andentrapment (cf. Example 16) of water-soluble antigens or membranefragments in muscle or skin to increase the efficiency of vaccination(use as adjuvant).

EXAMPLE 14

The following example shows that lecithin-coated microcrystals can beformed in the presence of a water-immiscible organic solvent in whichthe crystalline drug is not soluble. The example is based on the musclerelaxant dantrolene. Its physical form is bright orange crystals with amelting point of 279°-280° C. and with low water solubility. Dantrolene(7.9 mg) was added to a test tube, the following solvents were added(cumulatively), and the drug did not dissolve: 0.3 ml mineral oil; +0.4ml n-dibutyl ether; +0.4 ml methoxyflurane; +0.3 ml methoxyflurane; +0.3ml methoxyflurane; +0.3 ml mineral oil. The above was sonicated with themicrotip for 15 min. This resulted in a fine supension of dantrolenecrystals which sedimented in about 15 min. The mixture was resonicatedand 0.1 ml was removed and added to a test-tube containing 19.8 mgdilauryl phosphatidylcholine (lecithin). With swirling the lecithin waswetted but did not dissolve. Isotonic saline (1.5 ml) was added to thetube and the contents were sonicated. This resulted in a yellowishsuspension of the consistency and appearance of egg nog. After two daysstorage the contents of the tube separated into three layers which wereremoved individually from the tube. The bottom layer had a volume ofapprox. 0.05 ml was a yellow-reddish mass which was easily resuspendedin isotonic saline with gentle swirling. It contained the bulk of thedantrolene. It consisted of microcrystals of dantrolene wetted with theorganic solvents and coated with a layer of lecithin. The middle layer,which represented the bulk of the volume, was very turbid. The top layerwas lighter colored. The middle and top layers representedlecithin-coated microdroplets, as described by me in U.S. Pat. No.4,725,442 (1988). The microdroplets in the middle layer were richer inmethoxyflurane; the microdroplets in the top layer were richer inmineral oil. This example shows that if a stable, poorly-oil-solublecrystalline drug compound is selected, lecithin-coated microcrystalswill spontaneously form during sonication, even when organic solvent ispresent in large quantities. This example provided insight into thephysical interactions involved in the stability of thephospholipid-coated microcrystal.

EXAMPLE 15

This example shows how lecithin-coated microcrystal preparations of theanthelmintic drug albendazole can be diluted to give stable suspensionssuitable for administration in drinking water for poultry and cattle. Aconcentrated preparation (20% (w/v) albendazole, (w/v) 10% lecithin) wasmade as described in Example 7. An aliquot was diluted into 400 ml oftap water to give a 0.25 mg/ml suspension which was stored withoutagitation in a capped 500 ml sample bottle. Immediately after dilutionparticle size analysis was performed. It showed 10% of the material in254±200 nm particles, 85% in 2.7±0.5 um particles, and 5% in >3 umparticles. After 64 hrs, only 45% of the drug had settled to the bottomthird of the bottle. There was a thin translucent liquid film on thebottom. This was readily resuspended with a single inversion. Particlesize analysis showed 54% of the material in 17±11 nm particles, 13% in3.0±0.3 um particles, and 32% in >3 um particles. The test shows thatthe lecithin-coated microcrystal dispersed form is can be used inautomatic dilution (proportionator) systems, even in cases where flow isinterrupted for over 5 days.

EXAMPLE 16

This final example shows that the lecithin-coated microcrystal is auseful means of retarding the release of biomolecules after injectioninto tissue. Utility includes the sustained release of biologicals afterdepot injection and the prolonged retention of viral or bacterialantigen in the process of vaccination. Bovine serum albumin (BSA, 14-Clabelled) was taken as an example of a water-soluble biomolecule. TheBSA was admixed to a final concentration of 27 ug/ml with preformedoxytetracycline microcrystals (20% w/v OTC, 20% w/v) lecithin preparedas in Example 1. Laboratory rats were injected with 0.1 ml of theadmixture (a) intradermally or (b) intramuscularly, and the skin andmuscle injection sites were analyzed for 14-C BSA radioactivityremaining at sacrifice after two days. Controls were the sameconcentration of BSA in isotonic glucose solution and the sameconcentration of BSA admixed with lecithin vesicles (20% w/v) preparedby sonication. Table 6 shows that higher levels of 14-C BSA activity arefound in skin and muscle sites for the lecithin-coated OTC microcrystaladmixture.

                  TABLE 6                                                         ______________________________________                                        14-C BSA Activity Remaining in Tissue                                         2 Days After Injection                                                                   Glucose    Lecithin OTC                                            Tissue/Trial                                                                             Soln       Vesicles Microcrystals                                  ______________________________________                                        Skin 1     5.0%       3.0%     88.0%                                          Skin 2     4.4%       3.1%     17.5%                                          Skin 3     4.2%       3.9%     12.6%                                          Muscle 1   5.9%       0.0%     27.8%                                          Muscle 2   8.6%       8.2%      6.2%                                          ______________________________________                                    

These data suggest that the phospholipid-coated microcrystal can retainbiologicals and antigens in its interstitial aqueous space, decreasingtheir rates of release from the injection site and thus prolonging theiractivity. The usefulness of the coated microcrystal for administrationof biologicals or as a vaccine adjuvant (respectively) could beincreased by including an immunosuppressant or immunostimulant drug(respectively) in the microcrystal.

What is claimed is:
 1. A syringable, injectable pharmaceuticalcomposition consisting essentially of an aqueous suspension of solidparticles of a pharmacologically active water-insoluble drug substancein solid form, the solid particles having diameters of about 0.05 um toabout 10 um, coated with a 0.3 nm to 3.0 um thick layer of amembrane-forming amphipathic lipid which stabilizes the drug substancefrom coalescence and renders the drug substance in solid form acceptableto tissue of host.
 2. A syringable, injectable pharmaceuticalcomposition consisting essentially of an aqueous suspension of crystalsor solid particles of a pharmacologically active water-insoluble drugsubstance in solid form, the crystals or solid particles havingdiameters or maximal dimensions of about 0.05 um to about 10 um, coatedwith a 0.3 nm to 3.0 um thick encapsulating primary layer consisting ofcoating and enveloping layers of a membrane-forming amphipathic lipid,which stabilizes the drug substance from coalescence and renders thedrug substance in solid form less irritating to living tissue, and 25 nmto 3.0 um thick secondary layer consisting of a membrane-formingamphipathic lipid in vesicular form associated with and surrounding butnot enveloping the lipid-encapsulated drug particles, which compositionis substantially devoid of uncoated crystals or particles.
 3. Thecomposition of claim 1 or claim 2, in which the drug substance particleshave a diameter of about 0.1 um to about 3.2 um.
 4. The composition ofclaims 1 or 2, in which a water-soluble drug is rendered water-insolubleby complexation with a pharmaceutically acceptable compound producing acrystalline or solid form.
 5. The composition of claim 1 or 2, in whichthe weight ratio of drug to lipid is from about 1:1 to about 1,000:1. 6.The composition of claim 1 or 2, in which the membrane-forming lipid isa phospholipid.
 7. The stable, syringable aqueous suspension of thecomposition of claim 18 or claim 2, containing by weight from 0.01 toabout 40% of the drug substance.
 8. The stable, syringable suspension ofthe composition of claim 1 or claim 2, dispersed in a pharmacologicallyacceptable, water-miscible polar organic liquid with a dielectricconstant greater than 30, which polar organic liquid does notsubstantially dissolve the lipid membrane or membranes or the drugsubstance or a mixture of the polar organic liquid with water.
 9. Thecomposition of claim 2, in which the particles of drug substance arewetted with a water-immiscible oil of up to 0.25 gram per gram of drugsubstance, to facilitate contact between the drug crystals or particlesand the primary layer of amphipathic membrane-forming lipid, or to slowthe rate of drug dissolution or to otherwise modify the rate of drugrelease.
 10. The composition of claim 2, in which the particles of drugsubstance are precoated by a layer of a waxy phospholipid-compatiblesolid having a melting point between 37° C. and 100° C. before or duringthe application of the primary layer of membrane-forming amphipathiclipid, the waxy phospholipid-compatible solid selected from paraffin,tristearin, ethyl oleate, cetostearyl alcohol, cetyl alcohol, myristylalcohol, stearyl alcohol and petrolatum.
 11. A solid pharmaceuticalcomposition consisting essentially of the composition of claim 1 orclaim 2, devoid of water which, when water is added, gives an aqueoussuspension.
 12. The pharmaceutical composition of claim 1 or claim 2 ininjectable form for intravenous, intra-arterial, intra-muscular,intradermal, subcutaneous, intra-articular, cerebrospinal, epidural,intracostal, intraperitoneal, intratumor, intrabladder, intra lesionalor subconjunctival administration.
 13. The pharmaceutical composition ofclaim 1 or claim 2 in orally administrable form.
 14. The pharmaceuticalcomposition of claim 1 or claim 2 for topical application.
 15. Thepharmaceutical composition of claim 1 or claim 2 for inhalation.
 16. Thepharmaceutical composition of claim 1 or claim 2 for installation intothe eye.
 17. The pharmaceutical composition of claim 1 or claim 2 fordilution into drinking water.
 18. The composition of claim 1 or claim 2wherein the pharmacologically active water-soluble drug substance is apesticide.